Micriobiology Flashcards
BSC CLASSES:
In at front through HEPA to the outside or into the room through HEPA With possible contamination
BSC I
BSC CLASSES:
NO AIR RECIRCULATION; total exhaust to the outside
With possible contamination
I B2
BSC CLASSES:
70% recirculated, 30% exhausted
BSC II A1
BSC CLASSES:
same as II A1 but has 100 FLM (linear feet per min)
Vertical Laminar Flow: air flows in sheets.
BSC IIA or IIB are recommended in school/hospitals
BSC II A2
BSC CLASSES:
70% EXHAUSTED; 30% RECIRCULATED
Exhaust air outside the building; for radioisotopes, carcinogens, & toxic chemicals
Vertical Laminar Flow: air flows in sheets.
BSC IIA or IIB are recommended in school/hospitals
II B1
BSC CLASSES:
SUPPLY and EXHAUST air thru HEPA. Close cabinet for BSL-4 Research facilities
III
Certification of Biosafety Cabinet should be done after:
- Installation ;
- If Repositioned more than 18 inches ; or
- Annually, or as indicated by risk assessment.
PHYSICAL METHODS OF STERILIZATION: HEAT
MOIST:
Autoclave “Steam under pressure”
Tyndallization / Fractional sterilization
Inspissation
DRY HEAT:
Oven
121°C 15 psi for15-30 mins or 132°C 15 psi 30-60 min (for sterilization)
100°C 15 psi for 30 mins for 3 consecutive days (1st: vegetative, 2nd: spores, 3rd: remaining cells)
70-80°C for 2 hours 3 consecutive days (for heat sensitive, Lowenstein, Jensen)
160-170°C for 1.5-3 hours For drying; Glassware, cotton swab, oils,
petrolatum, powder
PHYSICAL METHODS OF STERILIZATION:
870-980degC
For research animals and medical/laboratory waste; safest
method to ensure no infective materials remain in samples
INCINERATION
PHYSICAL METHODS OF STERILIZATION: FILTRATION
Pulling liquids through a membrane (cellulose acetate or cellulose
nitrate) with a vacuum; For heat-sensitive materials like
antibiotic solutions, toxic chemicals, radioisotopes, vaccines, carbohydrates. Usual pore size: ___ Critical sterilizing for parenteral
solutions: 0.22 μm Filtering small viruses: 0.1 μm
has a 0.3um filtration; Used in isolation rooms, operating
rooms, biological safety cabinets
Membrane filtration; 0.45-0.80 μm
HEPA filter
PHYSICAL METHODS OF STERILIZATION:
Composed of short wavelength, high energy gamma rays
Used in sterilizing disposables such as plastic syringe,
vacutainer blood tubes, catheters, gloves
IONIZING/GAMMA
RADIATION
CHEMICAL METHODS OF STERILIZATION:
For heat-sensitive materials
Disadvantage: lengthy cycle times and health hazard
ETHYLENE OXIDE (ETO)
CHEMICAL METHODS OF STERILIZ ATION:
Oxidizing agent; Used in sterilizing HEPA filter in BSCs, metals, and
medical instruments like scissors. No toxic by-products
VAPOR-PHASE HYDROGEN PEROXIDE
CHEMICAL METHODS OF STERILIZATION:
H2O2 generates plasma by exciting the gas in enclosed chamber under deep vacuum with the use of radiofrequency or microwave energy
HYDROGEN PEROXIDE GAS PLASMA
PHYSICAL METHODS OF DISINFECTION: PASTEURIZATION
___: method 63degC, 30 min
___: method 72degC 15 sec Bailey’s: 70degC 30 min
kills food pathogens without
damaging the nutritional value or flavor. Properly pasteurized milk should have a ___ ALP/Phosphatase test.
Batch
Flash
negative
ANTISEPTICS:
Tincture of iodine: ___ + ___
70% ethyl alcohol followed by iodophor used in drawing blood culture and surgery.
___: Iodine + Neutral polymer (Povidone-Iodine, Poloxamer-
Iodine)
Contact time for must be >30seconds
Iodine must be removed completely from the skin to avoid irritation. Must be diluted properly. Not sporicidal
HALOGEN- Iodine
Iodine + Alcohol
Iodophor
ANTISEPTICS: PHENOLICS
Has high
antimicrobial activity better than iodine) and has low toxicity.
More effective on gram(+) than in gram(-)
Less effective in Fungi and Mycobacteria; Naked viruses are not inactivated.
Chlorhexidine gluconate (CHG)
Biological Indicator (Bioindicators) of Sterilization: Autoclave
Geobacillus (Bacillus) stearothermophilus
Biological Indicator (Bioindicators) of Sterilization: Dry heat/Oven
Bacillus subtilis
Bacillus atrophaeus
Biological Indicator (Bioindicators) of Sterilization: Radiation
Bacillus pumilus
Biological Indicator (Bioindicators) of Sterilization: Gas sterilant or
Ethylene oxide
Bacillus subtilis
Bacillus atrophaeus
Most commonly used autoclave:
Gravity Displacement Autoclave
most resitant to most sensitive order of microbes
Prions > Endospore > Mycobacteria > Naked/Nonlipid viruses > Fungi > Bacteria > Enveloped/Lipid viruses
Methods of Killing of Prions:
Autoclave: ___ in prevacuum sterilizer
__ for 1 hour in Gravity displacement sterilizer
1N NaOH: __ for 1 hour then autoclave
134degC for 18 minutes
132degC
Immersion
___: Single cellular except Cyanobacteria
2 Domains:
1. Bacteria (Environmental cyanobacteria/blue-green algae and
Medically relevant bacteria); and
2. Archaea (Extreme habitats) high salt conc, jet fuel, high temperature
Has circular chromosome; Cell wall has peptidoglycan
Prokaryotes (Prenucleus)
__:
Fungi and Parasites
Single or Multicellular organisms
Has linear chromosome
Cell wall has NO peptidoglycan.
Eukaryotes (True nucleus)
__:
Viruses - Host-dependent infectious agents
Prions – Abnormal infectious proteins
TAXA: order?
Infectious Particles
(do kings play chess on fine green silk)
Domain – Kingdom – Phylum – Class – Order – Family – Genus – Species
Classification, nomenclature, and identification of organisms
Deals with variations in gene expression not caused by nucleic acid sequence similarities or differences
It is a method of organizing microorganisms into groups or taxa
based on similar morphologic, physiologic, and genetic traits.
taxonomy
epigenetics
classification
Is the process consisting of a series of methods designed to provide the microbiologist with relevant and useful clinical information about a microorganism
Naming of microorganisms according to rules of International Code of Nomenclature of Prokaryotes (ICNP).
Is considered the same species with the same genetic make-up but displays differential physiologic characteristics.
identification
Nomenclature
Biotype / biovar
A strain of microorganism that can be differentiated based on antigen types using specific antibodies.
A specific version of an organism; a subtype of a specific organism with some minor differences
Characterization of etiologic agents using molecular techniques important during an outbreak or epidemic.
serotype/serovar
Strain
Strain typing
Macroscopic and Microscopic morphology, Staining characteristics, Environmental & Nutritional requirements, Resistance profiles, Antigenic and Subcellular properties, Chemotaxonomic properties
(teichoic acid, fatty acid, protein, cell wall composition)
DNA base composition ratio (guanine, cytosine, adenine, thymine), Nucleic acid base sequence (hybridization tests), Average nucleotide identity (ANI), Genome-to-genome distance
phenotypic criteria
genotypic criteria
the ability of the microorganism to produce disease in a host.
the degree of damage caused by the microorganism to the host; degree of pathogenicity.
growth of microorganisms that result in damage to host.
Ingestion of preformed bacterial toxins present in food.
Pathogenicity
Virulence
Infection
Intoxication
Infection is transmitted by direct contact between reservoir and host
Infection is transmitted to host via intervening agents (Vectors or Vehicles)
Results when infection produces notable changes in human physiology associated with damage or loss of function to one or more of the body’s organ system.
Direct transmission
Indirect transmission
Disease
A result of regular interaction with animals for food production
A larger than normal number of diseased or infected individuals that occurs over a relatively short period
Any type of epidemiologic investigation that involves data collection for characterizing circumstances surrounding the incidence or prevalence of a particular disease or infection.
Livestock-associated infection
Outbreak
Surveillance
Pioneers in the History of Microbiology:
Father of microbiology; 1st microbiologist, 1st to describe bacteria
phenol as antimicrobial
Father of Epidemiology
proposed heat in killing bacteria (aseptic technique) and pasteurization
Antonie van Leeuwenhoek
Joseph Lister
John Snow
Louis Pasteur
Pioneers in the History of Microbiology:
Father of Taxonomy
Germ theory; Anthrax & Tuberculosis
Father of Handwashing/Hospital hygiene
Endospore survival on boiling
Carl von Linne
Robert Koch
Ignaz Semmelweis
FerdinandCohn
Pioneers in the History of Microbiology:
Father of virology (discovery of virus); use of enrichment and selective media
Theory of chemolithotrophs; use of enrichment and selective media
Discovered HIV that causes AIDS
Developed >40 vaccines: against Hepatitis A and B, Measles, Mumps, Rubella,S. meningitidis, S. pneumoniae
Martinus Beijerinck
Sergei Winogradsky
Luc Montagnier
Maurice Hilleman
Pioneers in the History of Microbiology:
Developed Gram stain technique
Inventor of PCR
Petri developed petri dish
use of agar as culture media instead of gelatin
Hans Christian Gram
Karry Mullis
Julius Richard
Fanny Hesse
BACTERIAL STRUCTURE:
Cell wall
- Morphology determinant: cocci, coccobacilli, bacilli, curved, fusiform, spiral shapes
- Primary Basis of __
- Has PEPTIDOGLYCAN/______________ LAYER
- Composed of N-acetyl-D-glucosamine (NAG) & N-acetyl-D-muramic acid (NAM)
bacterial classification
MUREIN
GRAM POSITIVE
Outer membrane and Periplasmic space:
Peptidoglycan
Negative charge source
Flagella
Physical destruction
B-lactam antibiotic
Basic dyes
Absent
T H I C K
Teichoic Acid
2 Basal Rings
Resistant
Sensitive
Sensitive
GRAM NEGATIVE
Outer membrane and Periplasmic space
Peptidoglycan
Negative charge source
Flagella
Physical destruction
B-lactam antibiotic
Basic dyes
Present
Thin
Lipopolysaccharide (LPS)
4 Basal Rings
Sensitive
Resistant
Resistant
Most important DIFFERENTIAL staining test in Microbiology
Stains the microorganism’s cell wall.
Most critical step – ___
Iodine should be BASIC. Because dyes are ____________ /_____________.
GRAM STAIN
decolorizer
cationic/basic
Reagents of GRAM STAINING (mnemonic)
1° stain
Mordant
Decolorizer
2°Counterstain
(VIAS)
crystal violet
Gram’s iodine
95% alcohol/acetone
safranin
QC ORGANISMS FOR GRAM STAIN:
Gram Positive; Dark purple to Deep blue: ____
Gram Negative; Pink to Deep Magenta: ___
Staphylococcus aureus
Escherichia coli
Resists Decolorization: __ (4) (may appear as Gram-positive)
Appears as Gram Neutral/ Gram: __ (Poorly stained)
Poorly stained with Gram stain: (3)
Poorly stained with Safranin: (4)
NOT STAINED ___ (Chlamydia, Rickettsia, Ehrlichia, Coxiella), __
(Mycoplasma/Ureaplasma), for Dark-field microscopy (__)
Acinetobacter, Kingella, Psychrobacter, Methylobacterium
Mycobacteria (Poorly stained)
Campylobacter, Brucella, Tropheryma whipplei
Legionella, Gram negative anaerobes, Francisella
Intracellular; No cell wall; Spirochetes
__: Screening of quality is thru GRAM STAIN:
Bartlett’s classification, <10 epithelial cells, >25 pus cells under LPO
______________: (2x3 cm smear; 300 OIF is examined)
SPUTUM
DSSM
Demonstrates the presence of Mycolic Acid or Hydroxymethoxy Acid in bacterial cell wall. Confirms suspected TB cases if patient is symptomatic or CXR is suggestive
ACID FAST STAIN
Hot method: __
Cold method: __
BEST Routine AFS method: __
BEST Tissue AFS method: __
MOST Sensitive AFS method: __ (read 30 LPF)
Ziehl-Neelsen
Kinyoun
Ziehl-Neelsen
Kinyoun
Auramine-Rhodamine
Ziehl-Neelsen (MNEMONIC)
1° stain:
Mordant:
Decolorizer:
2°/Counterstain:
Acid fast:
Non-Acid fast::
(CHAM)
Carbolfuchsin
Heat
3% Acid Alcohol
Methylene blue
Red/Pink
Blue
Kinyoun
1° stain:
Mordant:
Decolorizer 3%:
2°/Counterstain:
Acid fast:
Non-Acid fast:
Carbolfuchsin
Phenol/Tergitol
3% Acid Alcohol
Methylene blue or Malachite green
Red/Pink
Green
Auramine-Rhodamine:
1° stain:
Mordant:
Decolorizer 3%:
2°/Counterstain:
Acid fast:
Non-Acid fast:
Auramine-Rhodamine
no mordant
0.5% acid
none
Yellow fluorescence
No fluorescence
Auramine-Rhodamine quenching agent ________; 3% Acid alcohol is composed of 3% HCl and 95% __.
AFB smear sensitivity: __
Sensitivity of Acid fast stain is 5,000 – 10,000 AFB/mL
Newly required number of sputum sample for TB detection: ___
Spot-Spot / Frontloading: 2 samples: __ apart
1. __: 2 samples; 1 random & 1 early morning sputum
~1/2 or 2/2 positive AFB sample → A confirmed case of tuberculosis.
0.5% potassium permanganate (0.5% KMNO4)
Ethanol
20-80%
2 sample
one-hour
Spot-AM / Spot-Early morning
CDC AFB Grading:
No AFB seen 0 AFB/__ fields
Indeterminate 1-2 AFB/__fields
1+ 1-9 AFB/__ fields
2+ 1-9 AFB/__ fields (at least 50 fields)
3+ 1-9 AFB/field
4+ __ AFB/field
300
300
100
10
>9
_______________ (Fading) – permanent loss of fluorescence due to chemical damage of fluorochrome
_______________ – a result of transfer of light energy to nearby molecules in the sample such as free radicals, salts of heavy metals, or halogens.
photobleaching
quenching
___ staining (urine) –M. smegmatis (Blue) vs M. tuberculosis (Red)
___ staining (tissue) – M. leprae (Red) vs M. tuberculosis (Blue)
__ stain (tissue) – for M leprae
Pappenheim’s
Baumgarten’s
Fite Faraco
__ for PARTIAL ACID-FAST microorganisms like Nocardia, Coccidians
Modified because they use __ such as 1% H2SO4 Alcohol.
2a. __
> Outer membrane
- found only in Gram __ bacteria
- Initial barrier of bacteria to the environment
- Composed of Lipopolysaccharide that gives the bacterial surface its ___ charge.
> Inner membrane
- A permeability barrier
- For cell wall synthesis
- site of __ synthesis as a counterpart of mitochondria
3a. __
- found only in Gram- negative; has gel-like substance that capture nutrients and detoxifies environmental solutes like antibiotics.
MODIFIED ACID-FAST STAIN
weak decolorizer
Cytoplasmic membrane
negative
negative
energy/ATP
Periplasm/Periplasmic space
- BACTERIAL DNA
A. dsDNA
B. __ – EXTRAchromosome that carries antibiotic resistance genes. - __
- __Bacteria; __ Fungi; for synthesis of proteins - CAPSULE
- consists of polysaccharide/carbohydrates except for __ (_______/amino acid,protein)
- contributes to bacterial colonization and __ in agar plates
- attachment and anti-phagocytic
__ = N. meningitidis, S. pneumoniae, K. pneumoniae, H. influenzae, Cryptococcus neoformans
Lab dx:___ reaction (Capsular swelling) and K or Vi Ag typing
PLASMID
RIBOSOME; 70s; 80s
B. anthracis; Poly-D-glutamic acid
slime layer/mucoid layer
ENCAPSULATED
Neufeld Quellung
___ – food reserve, lessens osmotic pressure
C. diphtheria:
Y. pestis:
M. tuberculosis:
Nocardia:
METACHROMATIC GRANULES
Babes-Ernst
Bipolar bodies
Much granules
Sulfur granules
- – for attachment (common/somatic pili) (N. gonorrheae, B. pertussis) and for gene transfer (sex pili, F factor)
Mechanism of Gene Transfer - Transformation:
- __ – Transposon (jumping genes), plasmid, sex pilus
- __ – Bacteriophage
PILI OR FIMBRIAE
Conjugation
Transduction
- __ – made of Calcium dipicolinate. Resistance to heat, chemicals, nutrient deprivation, drying.
Bacillus anthracis (__), Clostridium tetani (__), C. botulinum (__)
ENDOSPORE
center; terminal; subterminal
- __ – for locomotion
A-trichous: __ (Non-motile microorganism)
__-trichous: single flagellum located at one end of the cell
__-trichous: single flagellum located at both ends of the cell
__-trichous: group of flagella located at one end of the cell
__-trichous: multiple flagella located around the surface of the cell
→ __ – composed of Axial filament; for spirochete, cork-screw
FLAGELLA
no flagella
MONO
AMPHI
LOPHO
PERI
Endoflagella
type of motility?
Kingella, B. henselae
Listeria
Campylobacter
Vibrio
Treponema
Capnocytophaga
Motility: - Best observed at ______
TESTS FOR MOTILITY:
___ (Motility medium) – spreading (+)
___ – motility observed
Twitching
Tumbling
Darting
Rapid Shooting star
Cork screw
Gliding
room temperature
SIM
Hanging drop test
- __ – has thousands of enzymes and the site of protein synthesis
- __ – has the bacterial chromosome which is circular and complexed with RNA (Eukaryotic chromosome is linear and complexed with histones)
CYTOSOL
NUCLEOID
___ – are genomic regions in which virulence factors are encoded in.
___ – consists of monomicrobic or polymicrobic group of bacteria housed in a complex biochemical matrix. Adheres to implants and prosthetic devices; contributes to ___.
___ – Starved, Nutrient-deprived, slow-growing state of cells in a biofilm which displays reduced susceptibility to antimicrobial agents.
Mechanism of signaling: Gram negative: __ (AHL) and Gram positive: __
Pathogenicity islands (PAIs)
Biofilm; antibiotic resistance
Persister cells
Acyl homoserine lactone; Oligopeptides
__: S. aureus, CONS, Streptococcus, Listeria monocytogenes, P. aeruginosa, Acinetobacter, Aeromonas, Aggregatibacter spp., Salmonella spp., Enterobacter, Enterococcus, Escherichia coli, Klebsiella, Proteus, Serratia, Citrobacter koseri, and Candida albicans.
__ – are biochemically active substances that have particular effects on host cells
Biofilm producers
Toxins
___; a protein;
100degC heat stability: Destroyed;
Formaldehyde: Detoxified
Immunologic, convertible to toxoid vaccine
is Specific, Localized infection;
Kills host cells, interfere in cell signals and protein synthesis
EXOTOXIN: Gram-Positive
Protein-lipid-polysaccharide;
100degC: Intact, released from cell lysis
Not Detoxified by formaldehyde;
Non-immunologic, not neutralized by antitoxin;
Broad; Systemic infection; Either disrupt or form clots (DIC), Fever,
Activation of complement/immune system, Hypotension, Shock, Death
ENDOTOXIN: Gram-Negative
___: Immunodiffusion, PCR, Tissue culture, EIA, RIA, Limulus amebocyte lysate
➢ Horseshoe crab’s (Limulus polyphemus) blood cell called __ is separated from plasma and lysing them makes a “lysate”, when mixed with __, it will form clots/precipitates and cause
turbidity.
➢ Latex agglutination – for detection of toxins of __, E. coli O157, and __
Toxin testing
amebocyte
endotoxin
Clostridium difficile; Rotavirus
SPECIAL STAINS:
___ - Dyar, Victoria Blue, Calcofluor white (for Fungal chitin)
___ - Levaditi Silver Impregnation, Fontana tribondeau
___ - Toluidine blue
Cell wall
Spirochetes
Helicobacter pylori
SPECIAL STAINS:
___ - Ziehl-Neelsen, Kinyoun, Auramine-Rhodamine, Pappenheim, Baumgarten,
Wadefite, Gabett
___ - Neisser’s, Albert’s, Ljubinsky, LAMB (Loeffler’s Alkaline Methylene Blue), Burke’s
___ - Wayson’s
Acid Fast
Metachromatic granules
Bipolar bodies
SPECIAL STAINS:
___ - Dorner’s, Wirtz-Conklin, Schaeffer-Fulton
___ - Gray’s, Leifson, Caesares Gil, Fisher-Conn
___ - Anthony’s, Nigrosin, India Ink
___ - McFadyean
Endospore
Flagella
Capsule/Negative stains
Bacillus anthracis
SPECIAL STAINS:
___ - Acridine orange, Feulgen
___ - Gimenez, Macchiavello
___ - Dienes stain
Nucleic acid/DNA
Rickettsia
Mycoplasma
Schaeffer-Fulton stain, mordant, decolorizer and counterstain
malachite; heat; tap water; safranin
Bacteria’s dry weight is made out of 50% __ and 14% __.
__ – uses Carbon dioxide as sole source of carbon
A. __ – Use light as source of energy
B. __ – uses inorganic compounds as source of energy
___ – require complex substances for growth; requires organic substances for energy; (clinically significant bacteria that causes human infection)
Carbon; Nitrogen.
Autotrophs
Phototrophs
Chemolithotrophs
Heterotrophs
TEMP:
___ (0-20degC)
___ contamination (Food-Listeria) & Blood bank- __, P. fluorescens, S. liquifaciens
Psychrophiles/Cryophiles
Refrigerator
Y. enterocolitica
TEMP:
__ (20-40 degC)
Room temp contamination
Blood Bank - ___, B. cereus
Temperature enrichment method
A. __ enrichment (4oC)
B. __ enrichment (42oC)
Mesophiles
S. epidermidis
Cold
Hot
TEMP:
___ (40-60 degC)
Bacillus stearothermophilus
Room temperature (20-25oC) – BEST for ___ production; Use Sterile White swab to pick up colony.
Incubator temperature – ________; Fungal cultures - (__)
Thermophiles
Microbial Pigment
35-37degC; 30degC
pH:
Lactobacillus acidophilus
Also known as:
1. Doderlein bacilli (Vagina)
2. Boas Oppler bacilli (Gastrointestinal)
Vibrio spp.
Acidophilic <5.5 pH
Alkalinophilic >8.0 pH
Salt concentration:
__ – Salt-Loving bacteria (Requires NaCl for growth)
S. aureus – __ NaCl (MSA) Enterococcus – __ NaCl broth
Vibrio – 0, 3, 5, 8, 10% NaCl broth (All Vibrio are halophilic
except:____________________)
HALOPHILES
7.5%; 6.5%
V. cholerae and V. mimicus
21% O2; 0.03% CO2
Usual Incubator, AMBIENT air/ ROOM air
AEROBE
FUNGI, MTB, Brucella, Bordetella
ANAEROBE:
0% O2; 5-10% H2; 5-10% CO2; 80%-90% N2
Gas Pak Jar, AnaeroPack Palladium, Brewer jar, McIntosh & Fildes
Obligate anaerobe
Clostridium Bacteroides
ANAEROBE:
Do not require O2 but grows better with O2
Usual Incubator, AMBIENT air/ ROOM air
Facultative anaerobe
MOST pathogens, Enterobacteria
ANAEROBE:
Grows at the same rate with or without O2
Usual Incubator, AMBIENT air/ ROOM air
Aerotolerant anaerobe
Cutibacterium (Propionibacterium) acnes
5-10% O2 (decreased); 8-10% CO2(increased); 85% N2
Microaerophilic incubator or Jars
MICROAEROPHILE
Campylobacter. Helicobacter, Treponema, Borrelia
(sa camp konti oxygen sa loob, tas may ulcer, gumawa ng trap for boars)
5-10% CO2 (increased)
Candle Jar, Capnophilic incubator
CAPNOPHILE
Haemophilus, HACEK, Neisseria, S. pneumoniae
SPECIAL GROWTH SUPPLEMENTS:
___: X and V factors, IsoVitale X or Vitox
___: Cysteine and Iron
Haemophilus
Legionella
SPECIAL GROWTH SUPPLEMENTS:
__: Cysteine and Cystine
__: Cysteine, Methionine, Nicotinic acid
__: Cysteine and Vitamin B6 (Pyridoxal)
Francisella
Bordetella pertussis
Granulicatella, Abiotrophia
SPECIAL GROWTH SUPPLEMENTS:
__: Hemin and Vitamin K
__: Vitamin B1 (thiamine)
Anaerobes (Bacteroides)
B. anthracis and Trichophyton fungus
BACTERIAL GROWTH CURVE
Generation time: can be every __ (E. coli) to every __ (M. tuberculosis)
Phase sensitive to antimicrobials?
Phase that has decrease in nutrients, increase in toxins; where sporulation occurs?
30 minutes; 24 hours
Exponential/Log phase
Stationary/Plateau phase
CULTURE MEDIA. In general: pH: __, distilled/deionized water; Dissolved till clear and no particles seen.
Heat-labile or delicate media are sterilized through __ or through __ (pore size 0.2-0.45um)
Types of Bacterial Culture: __ (3)
NOTES: __ - most common solidifying agent
After the media has cooled from sterilization, just before dispensing, add supplements (blood, vitamins, nutrients, or antibiotics)
Dispense ___ of cooled (55-60oC) media on each 100 mm petri dish (commonly used).
7.2-7.4
116-118degC; membrane filtration
Pure culture, Mixed culture, Stock culture
Agarose
20-25 mL
Antimicrobials used in Culture media for Neisseria:
What inhibits Gram positive? negative? Fungi?
Vancomycin, Lincomycin, Colistin (VLC is nice)
Nystatin, Amphotericin B, Anisomycin
GRAM NEG MEDIUMS:
Inhibitory: Eosin Y, Methylene Blue
Indicator: Eosin Y, Methylene Blue
NLF: Colorless or Translucent
~ E. coli appears as ___ E. aerogenes as fish-eye colony
EMB (Levine agar); green metallic sheen
GRAM NEG MEDIUMS:
Inhibitory: Crystal violet, Bile salt
Indicator: Neutral red
NLF: Colorless
~__ – detection of E.coli O157:H7
MAC
Mac Sorbitol
GRAM NEG MEDIUMS:
Inhibitory: Bile salt
Carbohydrate: Lactose, Xylose, Dextrose
Indicator: Phenol red
LF: Yellow
NLF: Red/Colorless
~ Selective and differential for Salmonella and Shigella
XLD: Xylose Lysine Deoxycholate
GRAM NEG MEDIUMS:
Inhibitory: Bile salt, Bromothymol blue, Acid fuchsin
Carbohydrate: Lactose, Salicin, Sucrose
Indicator: Bromothymol blue
LF: Yellow
NLF: Blue Green/Colorless
~Differentiates Salmonella and shigella from other enteric gram-
negative bacteria
HEA: Hektoen Enteric Agar
GRAM NEG MEDIUMS:
Inhibitory: Bile salt
Carbohydrate: Lactose
Indicator: Neutral red
LF: Red/Pink
NLF: Colorless
~ For Salmonella and Shigella
DCA: deoxycholate citrate agar
GRAM NEG MEDIUMS:
Inhibitory: Brilliant green, bile salt
Carbohydrate: Lactose
Indicator: Neutral red
LF: Red/Pink
NLF: Colorless
~ For Salmonella and Shigella
SSA: Salmonella Shigella (SS) Agar
GRAM NEG MEDIUMS:
Inhibitory: Brilliant green
Carbohydrate: Lactose
Indicator: Bismuth sulfite
NLF: Black (Salmonella
~ For Salmonella
BSA: Bismuth sulfite agar
GRAM NEG MEDIUMS:
Inhibitory: Bile salt
Carbohydrate: Sucrose
Indicator: Bromothymol blue
LF: Yellow (sucrose)
NLF: Green (sucrose)
~ For Vibrio
TCBS: Thiosulfate–citrate–bile salts–sucrose agar
CULTURE MEDIA:
__: Fastidious and Non-fastidious microorganisms
__: For anaerobes, aerobes, microaerophilic and fastidious organisms
__: backup/enrichment broth for subculturing bacteria from primary agar plate
__: Fastidious and Non-fastidious microorganisms (for sterile samples)
__: Non-fastidious microorganisms
__: Fastidious organisms (Haemophilus, Neisseria, Brucella)
Nutrient agar
Thioglycollate broth
Trypticase soy broth
Brain-Heart Infusion Agar or Broth
Blood Agar
Chocolate agar
CULTURE MEDIA:
__: Each kind is intended for a specific bacteria like E. coli O157, MRSA, Candida albicans
__: Each kind is intended for “bacteria-specific” drug-resistant strains; Carbapenem-resistant, ESBL-positive, VRE, MRSA
__: Gram positive cocci and bacilli (aerobic/anaerobic)
__: Gram positive cocci
__: Staphylococcus aureus
Mueller Hinton agar with 2% NaCl or with 4% NaCl and 6 ug Oxacillin: for culturing ___
LIM, Todd-Hewitt broth, carrot medium, Granada medium; Hippurate broth (Identification purpose): for ___
Chromogenic media
Brilliance agar
Phenylethyl alcohol (PEA) agar
Columbia colistin-nalidixic acid (CNA) agar
Mannitol salt agar
MRSA
S. agalactiae
__: Group D streptococci, Enterobacter, Klebsiella, Serratia, Listeria
__: Vancomycin-resistant Enterococci
__: Enterococci
Polymyxin-Acriflavine-Lithium Chloride-Ceftazidime Esculin-
Mannitol (PAL-CAM broth): __
Cystine-tellurite blood agar, Loeffler medium, Potassium
tellurite agar, Tinsdale agar (TIN agar): __
Bile Esculin Agar (BEA)
Bile esculin azide agar with Vancomycin
Enterococcosel agar
Listeria monocytogenes
Corynebacterium
PLET agar (Polymyxin B-lysozyme-EDTA-Thallous acetate): __
MEYP/MYP; PEMBA; BCM: __
0.4% Dilute gelatin medium: __
Petragnani medium, Lowenstein-Jensen medium, Middlebrook 7H9 (broth): __
Selenite broth, Bismuth sulfite agar, Brilliant green agar: __
Phenylalanine Deaminase agar (PAD): __
Bacillus anthracis
Bacillus cereus
Nocardia and Streptomyces differentiation
Mycobacterium tuberculosis
Salmonella (the 2 agars are very selective for Salmonella)
Proteus-Morganella-Providencia (PMP group)
__: Yersinia enterocolitica, Aeromonas spp.
Cycloserine Cefoxitin Fructose Agar (CCFA): __
Egg yolk agar (EYA) AKA McClung Toabe agar: __
Cefsulodin-Irgasan-Novobiocin (CIN) agar
Clostridium difficile
Clostridium perfringens
Alkaline Peptone water (APW) and Thiosulfate citrate-bile salts
(TCBS) agar: __
__: Pseudomonas aeruginosa
Bordet-Gengou Agar, Regan-Lowe: __
__: agar with antibiotics Legionella
Wadsworth Brucella Blood agar:__
Blood agar with Kanamycin and Vancomycin (KV blood agar): __
Vibrio cholerae and Vibrio parahaemolyticus
Cetrimide agar
Bordetella
Buffered Charcoal Yeast Extract (BCYE)
Moderately-fastidious anaerobes
Bacteroides
TRANSPORT MEDIA:
Enteric pathogens (Stool samples): __
Neisseria gonorrhoeae and Neisseria meningitidis: __
Viruses: __
Amies and Stuart’s transport media: __
__: Chlamydia, Mycoplasma/Ureaplasma, and Viruses
Cary-Blair
JEMBEC media
VTM (viral transport medium
Aerobic and anaerobic bacteria
Universal transport media (UTM)
___ is done by conducting phenotypic and genotypic laboratory tests to gather information and identify the microorganism responsible for the disease. Before starting a work up, a PURE CULTURE must be used to yield a proper identification and then later on, susceptibility testing of the microorganism.
Work up/Identification Scheme
A. __: Use of slides in smear preparation, special staining, and manual biochemical testing.
B. __: Use of Analytical Profile Index (API) – for identification of fermentative gram negative bacteria. Has 20 cupules. __ profile number is recorded which is equivalent to a specific bacteria in the database.
C. ___: Colorimetry, Fluorometry, or Turbidimetry are used to detect bacterial growth.
Manual/Conventional Method
Manual Multitest System; 7-digit code
Automated Methods
Automated Methods:
MicroScan and TREK Diagnostic System: uses __
__: Uses 64 well cards for identification, sensitivity testing, and detection of multi-drug resistant isolates within 16 to 24 hours including anaerobic organisms. AST results are interpreted using ________________ (AES) within 6-8hours.
__: Through colorimetric and fluorometric system, a 136 well combination panels
Uses 96-well microtiter trays/plate
Vitek 2 System; Advanced Expert System
BD Phoenix
A. ___: Referred to as the gold standard for microbial typing especially those involved in nosocomial infection outbreak. Bacterial chromosome fragments are analyzed to
characterize each of the specie’s DNA profile.
B. ___: _ targets specific bacteria based on their surface receptors. A culture of bacteria is grown and inoculated with different phages. The positive result is characterized by lysis of colonies which means the specific bacteriophage has
successfully attacked the specific bacteria
Pulsed-Field Gel Electrophoresis
Bacteriophage typing
__: A DNA sequence that encodes a specific product
__: Comprises the all genes in an organism
__: Transcription and Translation
__: Site of active replication
Molecular assay: NEVER USE ___ SWAB with ____ shaft
gene
genome
Gene expression
Replication fork
calcium alginate swab; aluminum
POLYMERASE CHAIN REACTION (PCR) invented by __. It involves DNA Amplification or DNA Xeroxy. It is the most common nucleic acid amplification method used in molecular diagnostics. Conventional PCR has __ repetitive thermal cycles:
- Denaturation (_________) – extract nucleic acid with heat/chemical/enzyme.
- Annealing (_________) –Primer (specific to target DNA sequence) is added to the denatured DNA.
- Elongation (_________) – Taq Polymerase is used. Can amplify single copy into 10^7 to 10^8 copies.
Kary Mullis; 20-50
94degC
50-58degC
72degC
-A DNA or RNA segment extracted from the sample which serves as the target for PCR
-Oligonucleotide that is specific for the target sequence present in the template
-Synthesizes new strands of DNA
-AKA RNA-dependent DNA Polymerase, used ONLY in RT-PCR; Not present in other PCR assays. Synthesizes complementary DNA (cDNA) from an RNA template.
- Cofactor of DNA Polymerase
-Ensures proper pH for DNA Polymerase
-Used by DNA polymerase to synthesize new DNA strands during Elongation phase
-The instrument used in PCR which adjusts temperature depending on the step being conducted.
Template
Primers
DNA polymerase
Reverse Transcriptase
Magnesium Chloride (MgCl2)
Buffer
Deoxynucleotides (dNTPs)
Thermal cycler
Types of PCR:
1. __: Amplification is done followed by gel electrophoresis and band staining to detect amplified products
2. __: (Quantitative PCR or qPCR): Same PCR process of amplification, but accumulation of amplicons is detected real-time by fluorophores that sends fluorescent signals detected by the machine.
~ Reverse Transcription PCR (RT-PCR): __ is used instead of DNA. RNA is first converted into its complementary DNA (cDNA), then usual PCR steps proceed.
Conventional PCR
Real-time PCR
RNA template
Shaft of Swab:
Wooden: Toxic to _____________ and Mycoplasma
__: Used usually in a calcium alginate-tipped swab.
__: A polystyrene general purpose shaft for bacteria and viruses.
Chlamydia
Aluminum
Plastic
__ - Generally a poor specimen if aspirate or biopsy can be obtained.
APPROPRIATE for nasal/nasopharyngeal, oropharyngeal, eyes, ears, or genitals.
NOT FOR __ and __ !!!!
SWABS; ANAEROBES AND FUNGI
Tip of Swab:
__: Swabs that are for general use for bacteria and viruses because of excellent collection and release of samples.
__: Toxic to ______________________ (unless with charcoal).
__: Toxic to enveloped virus & Neisseria, and interferes on immunofluorescent and molecular (PCR) methods
Dacron & Rayon
Neisseria
Calcium Alginate
Design of Swab:
__: Can trap organisms, leading to decreased recovery/sensitivity
__: Uses Nylon fibers for enhanced uptake and release of microbes
Woven/Traditional
Flocked
Transport specimen in a leak-proof plastic bag with separation for paperwork MARKED with BIOHAZARD label. Most specimen should be transported __ to the lab with some few exceptions which are the sterile specimen (within __).
“__” the requirement of microorganisms suspected in a sample.
within 2 hours; 15 minutes
Maintain
SHIPMENT:
Triple packaging arrangement: ______________________
Sample > Primary container > Absorbent > Secondary container > Dry ice/Liq. nitrogen > Outer/Tertiary container
Category A: substances/microbes __ of permanent disability or life-threatening
Category B: substances/microbe __ of permanent disability or life-threatening on its current form
Double bagging technique
capable
incapable
PRESERVATION:
* Use __ for URINE (bacteriostatic for 3 days), Don’t use formalin or Polyvinyl alcohol (PVA) - For parasites only
* Stuart/Amies may contain _____________ to NEUTRALIZE FATTY ACID that is toxic to bacteria with pili
* Anticoagulants are used to prevent clotting of blood, bone marrow, synovial fluid (bacteria will be trapped therefore they’re not going to be isolated).
BORIC ACID
charcoal
__ – should be used since some anaerobes and Neisseria are susceptible to increased SPS concentration. can neutralzie with __
__ can also be used for viral culture, and Mycobacterium culture, although it inhibits some gram-positive bacteria and yeasts.
__, and others – should never be USED.
0.025% SPS; 1% gelatin
Heparin
Citrate, EDTA
Abscess, Lesion, Wound, Ulcer
Blood: Adults -preferred ___
Pediatric – preferred __
Bone marrow:
BAP, MAC, CAP; Deep abscess – use anaerobic media
≥ 20mL/set; 1-20 mL/set
Primary is in Blood culture. Subculture in BAP, MAC, CAP
BAP, CAP
Disinfect skin with __ followed by __. (pg 48, Bailey 15E; Ch. 8, Harr)
Draw blood during FEBRILE episodes. Draw __. DO NOT draw >4sets in 24hrs period.
ARD – Antimicrobial Removal Device
Manual - __ blood to broth ratio. Growth detected in the presence of pellicle/turbidity/hemolysis/bubbles.
Automated - __ blood to broth ratio. Growth detected by BacT/Alert (5days TAT)
70% alcohol; 2% iodine/iodophor/betadine
2-3 SET only per 24 hours
1:10
1:5
Amniotic, Peritoneal, Pericardial, Pleural fluids
Transport within 15 mins at __.
CSF/Spinal fluid:
In general, Transport: ________
Delayed testing: ______
Storage: __
Viral studies: __
~__ or use of __ for best sensitivity and organism recovery.
BAP, MAC, CAP, Anaerobic media (Bacteroides)
room temperature
22-25degC
20-24degC (Bailey’s)
35degC
4degC
Cytocentrifugation; acridine orange
Meningitis:
Newborn - __
Children - __
Adult - __
Geriatric - __
Immunocompromised (newborns, elders) - __
S. agalactiae, E. coli
H. influenzae
N. meningitidis
S. pneumoniae
L. monocytogenes
EAR:
Otitis externa:
__: S. aureus, S. pyogenes, __ (Swimmer’s ear/Acute diffuse otitis externa)
__: P. aeruginosa, Anaerobes
Otitis media:
__: S. pneumoniae, H. influenzae, M. catarrhalis, S. pyogenes, RSV, Influenza virus, coronavirus, enterovirus, rhinovirus
Chronic: __ (Bacteroides, Fusobacterium, Prevotella, Porphyromonas, Peptostreptococcus)
__ ear: BAP, MAC, CAP
__ ear: BAP, CAP, BBA
Acute: P. aeruginosa
Chronic
Acute
Anaerobes
External
Inner
Eye conjunctiva or Corneal scraping:
Transport within __ at room temp.
Corneal scraping is incubated at __ in __.
Direct inoculation to BAP and CAP (transport)
15 mins
28degC; SDA
__Gastric wash or lavage: usually for __.
For Mycobacteria detection, process within __ or neutralize with __.
Rectal swab: Insert 1-1.5cm deep, feces should be visible on swab. Inoculate in enteric media.
___ - all ages
___ - rotavirus
___ - - >3 days nosocomial diarrhea
MEDIA for stool and rectal swab: __ (3); for urine: only ___, ___
URINE (quanti counting): 1uL loop __: 10uL loop ___
infants or AFB
4 hours; 10% sodium carbonate
Norwalk – all ages
Rotavirus – children
C. difficile
BAP, MAC, Enteric media; BAP, MAC
1 colony x 1000 = CFU/mL
1 colony x 100 = CFU/mL
Urethra:
Swab in __. Collect __ after patient has urinated. culture media: __
__ – N. gonorrhoeae, Chlamydia trachomatis, Ureaplasma, Mycoplasma
Amie’s or Stuart’s medium; 1 hour; Thayer-Martin (culture)
Urethritis/Cervicitis
culture medias used for:
cervix, vagina and prostate
Intrauterine device (IUD): media?
Transport within __ at room temp. __ infections.
BAP, Thayer-Martin
cervix: +CAP
prostate: +MAC, CAP, CNA
THIO; 15 mins; Actinomyces
LEVELS OF SPECIMEN PRIORITIZATION:
Critical/Invasive specimen Needs immediate processing.
Amniotic fluid, Blood, Heart valve, Brain, CSF
Level 1
Nasal swab: culture media?
Specimens for viruses, chlamydia, and mycoplasma are usually submitted in appropriate transport media at __ (ice pack or liquid nitrogen).
MRSA agar; MRSA screening
4degC
LEVELS OF SPECIMEN PRIORITIZATION:
Unpreserved specimen. Needs to process after Level 1 samples to avoid overgrowth of normal flora or dying of fastidious organisms.
Bone, Wound Drainage, Feces, Sputum, Tissue,
Level 2
LEVELS OF SPECIMEN PRIORITIZATION:
Quantitation required (1:1000 or 1:100)
Catheter tip Urine (midstream/suprapubic/catheterized)
Level 3
LEVELS OF SPECIMEN PRIORITIZATION:
Preserved
Preserved Urine, Preserved Stool, Swabs in holding/ transport medium
Level 4
Cell wall synthesis inhibitor:
Cell membrane synthesis inhibitor: (binds and disrupts)
Nucleic acid synthesis inhibitor:
Inhibit RNA synthesis - __
Penicillin, Vancomycin, Bacitracin, Carbapenem (penny’s vase broken by carl)
Colistin & Polymyxin (g-); Daptomycin (for g+) (poor cats die/skibidap)
Sulfonamide and Trimethoprim (SXT), Nitrofurantoin, Nalidixic acid, Fluoroquinolones (-floxacin) and Rifampin
(sobrang tigas na niya, fuck rara)
Daptomycin, Linezolid, Tedizolid Streptogramin - for __
Colistin & Polymyxin - for __
All gram positive bacteria are __ to Vancomycin except some Enterococcus (VRE), Lactobacillus, Leuconostoc, & Pediococcus.
All gram negative bacteria are __ to Vancomycin except some Chryseobacterium, Moraxella, or Acinetobacter.
for G+ only
for G- only
susceptible
resistant
__: Reduced susceptibility of organism to a particular antimicrobial
__: Achieved when a particular antimicrobial can no longer be used clinically against a certain bacteria.
__: Antimicrobial resistance because of the microorganism’s normal genetic structural or physiological state
__: Antimicrobial resistance that results from the “altered” cellular physiology and structure caused by changes in a microorganism’s genetic make-up.
Biologic resistance
Clinical resistance
Intrinsic resistance
Acquired resistance
RESISTANT TO:
Anerobic bacteria:
Aerobic bacteria:
Klebsiella:
GN bacteria, Lactobacilli, Leuconostoc:
Aminoglycoside
Metronidazole
Ampicillin
Vancomycin
ANTIMICROBIAL SUSCEPTIBILITY TESTING
__ – TT1-TT5 without growth/clear, TT6 –with growth/turbid. This is the lowest concentration of an antimicrobial agent that prevents bacterial growth.
__ – broths after incubation are subcultured in agar, number of colonies are counted. This is the lowest concentration of the antimicrobial agent that actually kills the bacteria.
__ MacFarland Standard: __ of 1% H2SO4 and __ of 1.175% BaCl2
**A strict full 24 hours incubation is followed if testing for _________
**A strict __ incubator temperature is used if testing for MRSA because __ may mask methR
MIC (Minimum Inhibitory Concentration)
MBC (Minimum Bactericidal Concentration)
0.5; 99.5 mL sulfuric acid; 0.5 mL barium chloride
VRE, VRSA, MRSA
30-35degC; >35degC
AST:
INOCULUM SIZE: Broth Dilution vs Agar Dilution
__: REFERENCE METHOD; MIC is determined. Broth microdilution: 0.05 to 0.1 mL broth. Broth microdilution: ≥1 mL broth
__: Tests many organisms against 1 antibiotic. Can determine MIC for N. gonorrhoeae which is hard to be grown on broth dilution methods.
BROTH: 5 x 10^5CFU/mL
AGAR: 1 x 10^4 CFU/spot
broth
agar
1.5 x 10^8 CFU/mL
Mueller Hinton agar (MHA) is used
Add 2% NaCl to detect __; For H. influenzae use __; For N. gonorrhoeae use __:
MOST COMMON METHOD; Tests 1 microorganism against __ antibiotic
pH media: 7.2 – 7.4; Depth: __
Incubation time: __; 20-24hours if __ or __
Reading a Disk diffusion test:
Against a __ background, read with a reflected light (well-lit area). Read zone edges at the point where there’s no obvious growth.
Disk Diffusion/Kirby Bauer
MRSA; Haemophilus test medium; GC agar
many
4 mm
16-18 hours; Streptococci or Neisseria gonorrhoeae
dark
__: The specific concentration of antimicrobial that separate the different categories of susceptibility (Susceptible, Intermediate, or Resistant).
__: Antimicrobial agent that is considered the most sensitive indicator of drug mechanism
__: A cumulative susceptibility report that tracks resistance or susceptibility of
commonly isolated organisms to antimicrobials.
__: When there’s HEAVY GROWTH on wells with LOW ANTIMICROBIAL concentration
followed by 1 or more wells GREATLY REDUCED GROWTH (small button/light haze).
__: When there’s microorganism GROWTH on wells with HIGH ANTIMICROBIAL concentrations and NO GROWTH on 1 or more wells with LOW ANTIMICROBIAL concentrations.
Breakpoint
Predictor drug
Antibiogram
Trailing in broth dilution
Skipped wells in broth dilution
KIRBY BAUER:
Increase drying
Too thin media
Use of old cultures (>24h old bacteria)
<0.5 MacFarland used
Delayed in incubation
FALSE SENSITIVE
(large Zone)
KIRBY BAUER:
Increase moisture
Too thick media
>0.5 MacFarland used
Delayed application of disks
FALSE RESISTANT
(small Zone)
Hazes of Staphylococcus on Methicillin and Enterococci on Vancomycin zones should be read using __ light.
__ – ignore swarm, measure the defined zone of inhibition
Hazes with sulfonamide & trimethoprim testing – __ the haze, and measure zones
If no Proteus/sulfonamide/trimethoprim is used, and hazes are evident, do not ignore and investigate. Plates containing __ is read at the top of the open plate (lid is removed).
transmitted
Proteus Swarming
Ignore
blood
bac resitance: S. aureus, S. lugdunensis
5% Sheep BAP or MHA with 15 μg; ____________ (E) and 2 μg _______(Cd)
35degC, for 16-18 hours
bac resistance: S. pneumoniae, beta-hemolytic Streptococci
MHA with 5% sheep blood
35degC, 5% CO2, for __
__________________ (Report as Clindamycin Resistant)
D-zone / Inducible clindamycin resistance (ICR)
Erythromycin; clindamycin
20-24 hours
flattening of clindamycin zone
Modified Hodge test:
Enterobacterales __________ production __ like appearance, Indented or Enhanced growth
Cefoxitin:
to detect ________- mediated oxacillin resistance
bac resistance: S. aureus, S. lugdunensis and CONS resistance to __
Disk diffusion using MHA with 30 μg cefoxitin
(CNP) test - Confirmatory for Carbapenemase
carbapenemase
Clover-leaf
mecA
oxacillin
Carba NP
___ - determines the MIC of 1 antimicrobial against 1 bacteria
o _____/Epsilometer – 1 gradient of concentration of antibiotic in a plastic strip
o Liofilchem – 1 gradient of concentration of antibiotic in a paper strip
__ - is exhibited when a BACTERICIDAL antibiotic can only INHIBIT a bacteria (not
killed); most commonly associated to bacterial resistance to beta-lactam antibiotics.
Gradient Diffusion testing
E-test
Tolerance
__ – AKA Serum-bactericidal test (SBT), activity of drug using patient’s own serum.
- __– Bacteria is inoculated in various concentrations of antimicrobial, and incubated, and the plates are subjected to colony counting at different periods of incubation. The rate of killing thebacteria is measured over a specified period (Example: 6 hours, 12 hours, 24 hours incubation)
- __ – Identification of the effectiveness of multiple antimicrobial agents against a single bacterial strain.
Schlichter test
Time-Kill assay
Synergy test
Gram-Positive __
Family: Micrococcaceae
Genus: __
Characteristics: Gram-positive cocci, Catalase __, Colonizers
Cocci
Staphylococcus, Micrococcus
positive
Gram stain: Gram positive cocci in CLUSTERS
Aerotolerance: facultative anaerobe
Glucose Utilization: fermenter
0.04 U Bacitracin: resistant
Furazolidone: sensitive >/= 15mm
Modified Oxidase: negative
Lysostaphin: sensitive 10-16mm
Staphylococcus
Gram stain: Gram positive cocci in Tetrads/Sarcinae
Aerotolerance: aerobic
Glucose Utilization: oxidizer
0.04 U Bacitracin: sensitive >/= 10mm
Furazolidone: resistant
Modified Oxidase: positive
Lysostaphin: resistant
Micrococcus
Colonizers of Anterior nares, nasopharynx, perineal area, skin, mucosa
Inhibit phagocytosis: Capsule, Slime layer, Biofilm
__: Affinity to Fc of IgG and complement
Exotoxins:
__ – disrupt smooth muscle in blood vessel, toxic to RBC, WBC, platelets, & hepatocyte
Beta – work with __ toxin, sphingomyelinase that hydrolyses cell membrane (cell lysis)
__ – cytolytic to RBC
__ – works with Panton-Valentine Leukocidin;
__ toxin
__ disease: Scalded Skin, Syndrome/Ritter disease, Pemphigus neonatorum
Culture:
BAP: Medium-large ____ convex, opaque __, _-hemolytic
MSA: __ colonies & halo; CHROMagar: MRSA is __-color; PEA, CNA, MSA: for stool
Positive: __
Negative: __
PCR: __ gene (ID of S. aureus), __ gene (ID of MRSA)
Staphylococcus aureus
Protein A
Alpha
alpha
Delta
Gamma
Exfoliatin
Exfoliative
pinhead smooth
creamy yellow
β
Yellow
mauve
VP, Slide and Tube, Coagulase!!!, DNase!!!
PYR, Microdase
nucA, mecA
COCCIS:
Inhibit phagocytosis: Slime layer, Biofilm, Delta exotoxin
Nosocomial bacteremia from indwelling vascular catheters, endocarditis from prosthetic heart valve, infections from prosthetic joints, etc.
Coagulase-Negative!!! (also S. saprophyticus) 5 μg Novobiocin – Sensitive
Seen in sexually-active young women urinary tract infection
Staphylococcus epidermidis
S. saprophyticus
Tests for Micrococcus and Staphylococcus:
Screening test for Gram-positive and differentiates Micrococcaceae from Streptococci (+ is __)?
Differentiate Micrococci (+ is color __ in M. luteus) from Staphylococci
Differentiate S. aureus from CoNS: 2 types? positive results in S. aureus?
Distinguish S. aureus, Serratia, and M. catarrhalis? 2 mediums used? reagents used for each? what is (+) result in S. aureus? (-) result in E. coli?
Catalase test; effervescence
Microdase (Modified Oxidase) test; Blue to Purple-blue
Coagulase test; tube and slide method
SLIDE: clumping; TUBE: clot
DNase test; DNase agar and agarose slant;
agar: methyl green; slant: toluidine blue and HCl
(+) agar: solorless/clearing; slant: deep pink or DNA precipitates
(-) agar: green; slant: blue or no ppt
Aids in ID of some GP cocci and bacilli. What is the (+) result (mnemonic) and (-)?
Differentiates Clumping factor positive Staphylococci? what is the (+) result for S. intermedius and lugdunensis? in S. aureus?
Voges-Proskauer (Coblentz method)
BaBasa Lang Saglit: (RED) B. anthracis, B. cereus, S. aureus, L. monocytogenes, Streptococci anginosus; (-): Yellow
PYR test; bright red; no color change
Tube coagulase detects? slide coag?
where are they found? temp needed for each?
who are the CoNS? (mnemonic)
free coagulase; cell-bound coagulase
S: surface of cell T: bacterial suspension
S: RT; T: 35degC for 1-4 hours
SHEWL: S. saprophyticus, haemolyticus, epidermidis,
Loeffler Serum Slant:
Golden yellow
Lemon yellow
Porcelain white
Characteristics: Gram-positive cocci in pairs or chains, Catalase and oxidase negative, Facultative anaerobe Classified according to Rebecca Lancefield grouping except S. pneumoniae and Viridans
S. aureus
S. citreus
S. albus (S. epidermidis)
Streptococcus, Enterococcus, and similar organisms
Streptococcus, Enterococcus, and similar organisms:
_____:
Pyogenic - Grows at 37degC only; ___
Viridans: Grows at 37degC & 45degC; __
Lactic: Grows at 10degC & 37degC; __
Enterococcus: Grows at 10degC, 37degC, & 45degC; __
Academic/Bergey
S. pyogenes, S. agalactiae
S. mutans, S. mitis
S. lactis
E. faecalis
Streptococcus, Enterococcus, and similar organisms:
Smith and Brown:
Alpha-hemolytic:__
Beta-hemolytic: __
Gamma-hemolytic: __
S. pneumoniae
S. pyogenes, S. agalactiae
S. bovis
Streptococcus, Enterococcus, and similar organisms:
Lancefield: (mnemonic)
Group A:
Group B:
Groups C, G:
Group D:
PADEE
S. pyogenes
S. agalactiae
S. dysgalactiae, S. equi
Enterococci
(Group A) Streptococcus pyogenes:
___– Responsible for 2 sequelae and binds Factor H; can cause what?
___– Epithelial cell attachment (Fibronectin binding)
DNase; Streptokinase; Hyaluronidase; ___ – Scarlet fever
Taxo A: __; PYR: __; ASO titer; ___; Schulz-Charlton test
STREP O vs S:
M protein; Rheumatic fever (Class I) & Acute Glomerulonephritis (Class I or II)
Protein F
Pyrogenic exotoxin
Sensitive; Positive; Dick’s test
O: Oxygen labile, Antigenic, Subsurface hemolysis (stab)
S: Oxygen Stable, Non-Antigenic, Surface hemolysis (streak)
GROUP AND SPECIES?
VF: Capsule, Activation of complement
LIM, Todd-Hewitt, Granada, Carrot medium
VF: Endogenous strain of skin, nasopharynx, GI, and genital that may gain access to sterile site; β-Hemolytic
VF: Biofilms, Adhesins (medical implants), Cytolysin, Hemolysin, peptide inhibitors
Nosocomial or Healthcare-associated; Enterococcosel agar, Positive in Bile esculin, 6.5% NaCl tolerance, and PYRase
(Group B/GBS) Streptococcus agalactiae
(Group C) Streptococcus dysgalactiae subsp. equisimilis and other group A, C, G, L
(Group D) Enterococcus faecalis
Enterococcus faecium
Normal flora of nasopharynx;
VF: Capsule – Pneumolysin!!!, mediate oxidative outburst in phagocytosis
Secretory IgA protease
Phosphorylcholine – Spreading and invasion
Causes: Meningitis, CA-Pneumonia, Otitis media,
Microscopic : Encapsulated __ shaped diplococci
Macroscopic: __-hemolytic, mucoidal, __ colonies
__ Sensitive, Bile solubility, Neufeld quellung
Streptococcus pneumoniae
bullet/lancet
Alpha; umbilicate
Optochin
(bullet sa head, kita umbilical cord, alpha male, may suka na green sa chin)
Normal flora of oral cavity, GI tract, female genitals
Alpha hemolytic, __ smell on CAP, __ positive (pink color), PYR negative
Viridans Streptococci
Butterscotch; LAP
Tests for Streptococcus:
Presumptive test for S. pyogenes: (2)
Presumptive test for Enterococci and Streptococcus bovis; Organisms are capable of GROWING in __ and hydrolyzing it to esculetin; Reagent: Ferric ammonium citrate (Iron); (+) ___ ppt
Presumptive test for S. pneumoniae, differentiates it from other alpha-hemolytic Streptococci? reagent for the BST is 10% or 2% Sodium deoxycholate (+) result on S. pneumoniae
For Enterococci; reagent: Cinnamaldehyde; (+) result on E. faecalis? (-) result on Aerococcus viridans
Bacitracin susceptibility (BAP) and PYR test (+) is bright red
Bile Esculin Hydrolysis; 40% bile; growth AND blackening (negative if one only)
Optochin (P disk) susceptibility test and Bile solubility test; Colony in agar disintegrates or Broth culture becomes clear.
LAP test; Red; Yellow/No color change
Tests for Streptococcus:
Differentiate Group B streptococcus from other Streptococci; (+) result on S. agalactiae? (-) result on S. pyogenes?
Presumptive ID of some organisms; reagent: 0.2 mL ninhydrin reagent; (+) result on S. agalactiae; (-) result on S. pyogenes
Tests for Enterococcus, faecalis (+) vs Enterococcus faecium; reagent: Bromothymol blue; (+) result on E. faecalis; (-) result on S. gallolyticus
For Enterococci’s ability to withstand high salinity; reagent: Bromocresol purple; (+) result on E. faecalis; (-) result on S. gallolyticus
CAMP test, arrowhead hemolysis; no enhanced/arrowhead hemolysis
Hippurate hydrolysis; deep purple; colorless or yellow pink
Pyruvate test; Yellow; No change or Yellow-Green/Weak
Salt Tolerance test; Growth & Yellow; Little/no growth
General characteristics: Gram positive bacilli, Aerobic, Spore-forming
Category A Bioterrorism Soil-isolate contracted by animals (Exposed industrial raw worker, military, public health personnel)
VF: Capsule; TOXINS: __ and __; toxins are made of __ as its factor
DSE: Cutaneous anthrax (or___): Pulmonary/Inhalational anthrax (__): Gastrointestinal or Ingestional anthrax (_): Injectional anthrax:
Gray, Flat, __, comet-tail/__ or ground glass colonies in BAP; Large Gram positive bacilli in chains resembling __
Spore-inducing techniques: Growth in TSI, Urea, NA with Manganese sulfate, or bicarbonate medium
Heat shock (degC and mins?)
Selective media: __; Presumptive test: 10 U Penicillin susceptibility test (__ appearance), PCR (__ capsule gene)
Bacillus spp.
Bacillus anthracis; Lethal toxin (LT), Edema toxin (ET); Protective antigen (PA);
Black eschar;
Woolsorter’s or Rag-picker’s disease:
Toxemia and sepsis.
Nonhemolytic, Medusa-head, bamboo-square
62-65degC for 15-20mins.
PLET agar; String of pearl; capBCA
Blood bank contaminant:
VF: Emetic type is Heat-stable, acid- resistant Cereulide enterotoxin
Diarrheal type is Hemolysin BL (HBL), Nonhemolytic enterotoxin (Nhe), Cytotoxin K (CyK AKA hemolysin IV)
Large, feathery, spreading __ on BAP, Wide zone of clearing in __________; Culture media: MEYP/MYP, PEMBA, BCM (anything egg agar); Heat shock: 70degC for 30mins. or 80degC for 10mins.
Ingestion of toxins in rehydrated foods; same appearance on BAP with B. cereus; toxins are: Cytotoxin _, enterotoxin
Bacillus cereus
Beta hemolytic; egg yolk agar
Bacillus cytotoxicus; K
Cereulide toxin is used in insecticides and pesticides: __
eye infection among drug users: __
B. subtilis is large, ___, ___, ground- glass, __-hemolytic, and may be pink/yellow/orange pigmented colonies on BAP
B. thuringiensis – Large, __, spreading __ hemolytic on BAP; Parasporal crystals/Insecticidal proteins
Large, moist blister colony with ameboid spreading in young cultures while wrinkled old cultures that is non-hemolytic seen in BAP
B. thuringiensis
B. subtilis
flat, dull; beta
feathery; Beta
Paenibacillus polymyxa
B. anthacis vs B. cereus:
wide-zone lecithinase, capsule, motility, hemolysis, gelatinase, penicillin G?
B. anth: +, +, -, -, - sensitive
B. cer: +, -, +, +, +, resistant
___:
General characteristics: Gram positive bacilli, Non-spore forming bacilli __ are short-slightly curved rods with rounded ends arranged in palisades
Not part of Normal flora. Nasopharynx in carriers. VF: Diphtheria toxin (inhibits protein synth)
Biotypes:
1. Intermedius – _______, gray, translucent β-hemolytic colony
2. Mitis – ______-sized, convex, smooth, black pigment fried-egg appearance, β-hemolytic colony
3. Belfanti – medium-sized, grayish-black, opaque, β-hemolytic colony
4. Gravis – ______, flat, glossy, grayish-black, daisy-head appearance, __-hemolytic colony
STRAINS: toxigenic is respiratory diphtheria; non-toxigenic is Cutaneous diphtheria or Velat sore/Barcoo rot
Presumptive: Brown-Black colonies with gray-brown __ in __ agar
Nutrients: Cystine; Culture: Tinsdale, __ medium (No ↑CO2)
Toxigenic strain ID: _______immunodiffusion test, Tissue culture, EIA, PCR, Schick skin test, Roemer test (Guinea pig virulence test)
Corynebacterium spp., Listeria spp., Erysipelothrix spp., and the likes
Corynebacterium
Corynebacterium diphtheriae
small; medium; large; Non
halo; Tinsdale; Loeffler; ELEK;
Zoonosis: cattle (Summer outbreak); Urease & Reverse CAMP (+); Glycogen fermentation (+),
Zoonosis: sheep, goats, horses; causes Suppurative
granulomatous lymphadenitis; Urease & Reverse CAMP (+); Glycogen fermentation (-),
Colonizer of Male genitourinary tract; Positive CAMP, glucose and sucrose fermentation
Corynebacterium ulcerans
Corynebacterium pseudotuberculosis
Corynebacterium glucoronolyticum
Normal flora of skin of hospitalized patients in inguinal, axillary, or rectal sites or from Intravenous catheters; Multiple antibiotic resistance in hospital setting; seen in indwelling catheters, prosthetic heart valves, CSF shunts
Enhanced growth with lipids (lipophilic): Add __ or Serum in media; __ on Nitrate and Urease
Normal flora of skin; same as above in medium; Struvite crystals in urine, Rapid urease +
Normal flora of skin; causes ___ or superficial, chronic, pruritic reddish-brown macules in skin. Lesions are __ fluorescence under a Wood lamp
Corynebacterium jekeium;
Tween 80; Negative
Corynebacterium urealyticum
Corynebacterium minutissimum; Erythrasma; coral red
Colonizers in animals, soil, vegetable. Sources: Meat, processed foods, soft cheese, vegetables, dairy products like milk, or colonized mother to fetus.
VF: __ - pore-forming toxin, hemolytic, cytotoxin that reduces responsiveness of T-cells; __ – Eats iron from transferrin; __ – Escape WBC and spread in blood; __ – placental invasion; __ – induce phagocytosis; __ – causes actin polymerization resulting to pseudopod projections causing cell-to-cell infection
DSE: Neonatal Listeriosis, Granulomatosis infantisepticum
Short gram-positive bacilli that may occur in singly or short chains
__ motility when incubated at NB at RT for 1-2 hours. ___ growth or ___ in SIM at 22-25degC. __ enrichment: Specimen in 4oC for
weeks-months
Selective media: PAL-CAM, McBride medium; __ test - purulent
conjunctivitis in rabbit; __ in Catalase, CAMP (__-type hemolysis), Voges- Proskauer, Esculin;
__ on H2S, Urease
Listeria monocytogenes
Listeriolysin O; Siderophores; Phospholipase; E-cadherin; Internalin; Act A
Tumbling; umbrella-like; inverted Christmas tree; Cold
Anton; Positive; Block
Negative
Zoonosis of veterinarians, meat (pork) butchers, and fish handlers; Normal flora
VF: Capsule, Neuraminidase, Hyaluronidase, Surface proteins
DSE: ___, Bacteremia
Gram positive short bacilli forms long filaments __-hemolytic on bap
__ on catalase, oxidase, esculin, nitrate, urease; __ in TSI. Test-tube brush/Pipe cleaner appearance in gelatin stab culture at 22degC
Curved Gram-positive rods with pointed ends and rudimentary branching, Reverse CAMP positive
Normal flora of vagina, GI tract; Pleomorphic (Long chaining bacilli, coccobacilli, spiral forms); __-hemolytic on BAP
Erysipelothrix rhusiopathiae
erysipeloid
Alpha; Negative; (+) H2S;
Arcanobacterium haemolyticum
Lactobacillus acidophilus; mouthAlpha
Actinomcyetes WITH Mycolic acid on Cell wall;
Acid-fast (underlined):
Partial acid-fast (not underlined):
Nocardia, Rhodococcus, Gordonia, Tsukamurella, some Corynebacterium
Lawsonella, Segniliparus
Cell wall peptidoglycan has Diaminopimelic acid (DAP), Arabinose,
and Galactose; In agar: Substrate hyphae and Aerial hyphae
__ appearance, with hyphae. Partial Acid Fast, __, __ & __ positive. LYSOZYME Resistant; Extensive Hyphae In __.
Culture media: SDA, LJ, BHI, BCYE, MTM – Incubate at __ for 2-3 weeks at __. Sulfur Granules from specimen emulsified in 10% KOH, crushed, and Gram stained: Small yellow-orange __ shaped with __-like structure
__ – most common Nocardia isolate.
NEGATIVE CASEIN, Xanthine, Tyrosine Hydrolysis
__: positive in Casein & TYROSINE Hydrolysis
Nocardia spp.
Beaded; Strictly aerobic; Catalase & Urease; Tap Water Agar
30degC; ambient air; kidney; club
Nocardia asteroides (sensu stricto)
Nocardia brasiliensis
Mucoid orange to red or Salmon-pink non-hemolytic colonies
Gram positive coccobacilli with “zigzag” scanty branching pattern
Positive in Urease, Catalase, & CAMP
Rhodococcus equi
Glabrous Waxy heaped gray-white colonies
Gram positive atypical bacilli with “spore-like bodies” and extensive branching that do not fragment
Musty basement odor
Extensive Hyphae In Tap Water Agar - LYSOZYME Sensitive
Streptomyces spp.
Gram positive actinomycete
Gold standard testing: PAS stain in gastrointestinal biopsy
First identified via PCR of duodenum biopsy
causes: __
Tropheryma whipplei
Whipple’s disease
General characteristics: Acid-fast bacilli (with Mycolic acid), Aerobic, Agents of pulmonary and skin diseases
__ is seen mostly in the poor, homeless, IV drug users, alcoholics, & the elderly.
__ – Productive cough, hemoptysis, Low-grade fever, night
sweats, anorexia, weight loss. In CXR, formation of Gohn lesion with
“granulomas/hard tubercles”, giant cells (cellular fusion with multiple
nuclei). Spread is through lymphatic or blood causing Meningeal/Miliary/Disseminated tuberculosis (common in
immunocompromised).
__ - precedes with no S&S, no active disease, and not transmissible. Pott’s disease or tuberculosis spondylitis/skeletal tuberculosis – __
Mycobacterium spp.
Tuberculosis
Pulmonary TB
bone TB
Biosafety Class:
__: Acid fast smears and culture for NON-aerosolizing procedures.
__: Manipulation with Aerosolizing procedures, Opening centrifuge vial, adding reagents to biochemical tests, molecular tests, and sonication. After aerosolizing procedures, stand specimen for 15 minutes.
Biosafety Level 2
Biosafety level 3
M. tuberculosis vs M. bovis?
Niacin Nitrate reduction Pyrazinamidase Catalase Urease T2H/TCH
Specimen:
Sputum (DOH, __ samples; Bailey __ samples – each are 8 hours apart),
__: (specimen of choice for <12 years old), 3 consecutive First-morning midstream clean catch urine, Stool (AIDS, M. avium complex), Tissue samples (incubated at 35oC and 30oC), SPS, Heparin, or Citrate Blood culture, and CSF.
M. tub: positive all; catalase neg; resistant to T2H/TCH
M. bov: negative all; T2H/TCH senstive
2; 3
Gastric lavage
Digestion-Decontamination methods (FOR TB):
1. __ – 2-4% NaOH, both decontaminant and digestant.
2. Zephiran-Trisodium phosphate method
3. __ – most commonly used for TB specimen processing; used in MGIT 960.
Other reagents used in TB:
1% Cetylpyridinium chloride – prolongs shelf life if sputum sample for up to __
__ – Decontaminant for thin samples like Urine.
__ – used for patients with cystic fibrosis (M. abscessus)
__ specimen: Centrifuge and use sediment for microscopy and culture
__ specimen: Liquefaction → Decontamination → Neutralization → Centrifugation → use sediment for microscopy and culture
NaOH method.
NALC-NaOH method
8 days
Sulfuric acid
Chlorhexidine
Sterile
Nonsterile
Culture media for TB:
A. Solid Media
1. Egg-based/Opaque media: __ and __; Lowenstein-Jensen, American Thoracic Society (ATS) Medium, Petragnani medium, Wallenstein medium. MYCOBACTERIUM TUBERCULOSIS COMPLEX
2. Serum-based/Transparent media: __ 7H10 and 7H11 and __ 7H11. __ is for protection of AFB against toxins. Casein hydrolysate for Isoniazid-resistant MTB
B. Liquid Media
1. BACTEC 12B: used for Mycobacteria Growth Indicator Tube (MGIT 960) system
2. Middlebrook 7H9 broth and Dubos Tween Albumin broth
C. Biphasic Media
__ AFB – mixture of TB culture media for rapid identification of mycobacteria
Automated methods for Mycobacteria
1. BACTEC 460 TB System:
2. Mycobacteria Growth Indicator Tube (MGIT 960) system:
3. BacT/Alert MB
4. versaTREK culture
has Egg yolk and Malachite green
Middlebrook; Mitchison; Albumin
SeptiCheK
Immunologic Tests for TB:
uses skin hypersensitivity to induce erythema and induration; Poor sensitivity, & anergy on patients with HIV.
MEDICATIONS:
__/__ Anti-Tubercular drugs: Rifampicin, Isoniazid, Pyrazinamide, Ethambutol
__: Resistant to at least two first line anti-TB drugs.
__: Resistant to Isoniazid, rifampicin, fluoroquinolones, and at least one of the 5 second-line TB drugs (Amikacin, Amithiozones, Bedaquiline, Ethionamide, or Capreomycin).
Mantoux/Tuberculin/PPD
First-line/Primary
MDR-TB
XDR-TB
Nontuberculous Mycobacteria characteristics:
Runyon Group?
Classification: Photochromogens, Scotochromogens, Non-Photochromogens. what are their growth rate? color when dark or exposed to light
Rapid Growers’ Runyon grp? growth rate?
phot: GROUP I
scot: GROUP II
non: GROUP III
10-21 days; Cream/Buff for dark, Yellow to Orange after light exp
GROUP IV; 3-7 days
Nontuberculous Mycobacteria: GROUP I
Fresh water and Salt water; Fish with contaminated water or breaks
on skin; __ Tween 80 Hydrolysis, Urease, Pyrazinamide
DSE: Cutaneous disease (__), bacteremia; Optimum growth temperature: __
In tropical environmental water (primarily in Australia);
DSE: Pulmonary disease, bursitis, tenosynovitis
Found in tap water, More common in white Males __, Catalase, Tellurite Reduction, & Tween 80 Hydrolysis
DSE: Chronic Pulmonary disease, cervical lymphadenitis, cutaneous disease
M. marinum; Positive; Swimming pool granuloma; 28-32degC
M. asiaticum
M. kansasii; positive in nitrate
M. branderi
other species in GRP I: M. intermedium and M. nebrakense
Nontuberculous Mycobacteria: GROUP II
Fresh/Tap water (pipeline, laboratory faucet) Positive Catalase and Tween 80 hydrolysis;
Raw milk, dairy products, soil; causes cervical adenitis in children
contaminants in water and soil
M. gordonae
M. scrofulaceum
M. szulgai
other species under GRP II:
M. europaeum, M. interjectum, M. heckeshornense & M. lentiflavum
Nontuberculous Mycobacteria: GROUP III
Environmental isolate; Positive Tellurite reduction and Pyrazinamidase; DSE: causes Johne disease in cattle, sheep, goats; Patients with AIDS: Disseminated disease
Immunocompromised: coinfection with HIV
Disseminated disease in AIDS patients
Hot water taps in hospitals; Optimum growth temperature: 28-32oC
Stagnant topical waters, aquatic insects; Positive heat-stable catalase; can cause African Buruli ulcer or Australian Bairnsdale ulcer
M. avium complex (MAC)
M. celatum
M. genavense
M. xenopi
M. ulcerans
other species in GRP III:
M. shimoidei, M. simiae, M. terrae complex, M. lacus, M. malmoense & M. haemophilum
Nontuberculous Mycobacteria: GROUP IV
__% pulmonary disease caused by this group; it is cystic fibrosis-associated
seen in environmental, Water, soil, dust
M. abscessus; 80%;
M. fortuitum and M. chelonae
RGT: 30% H2O2 and 10% Tween 80
(+): >45mm of bubbles (M. flavescens)
(-): <45 mm column of bubbles (M. tuberculosis)
RGT: same as above + water bath
(+): Bubble formation (M. flavescens)
(-): No change (M. tuberculosis)
RGT: Nutrient broth with 1% Potassium nitrate; Indicator: Sulfanilic acid
(+): red (M. tuberculosis); (-): No color change (M. marinum)
RGT: Lowenstein Jensen with ≥50 colonies; (+): Yellow
(M. tuberculosis); (-): No color change (M. fortuitum)
RGT: Tween 80; Indicator: Phenol red
(+): Pink color in 10 days (M. kansasii); (-) No color change (M. intracellulare)
Semi-Quantitative Catalase test (SQC)
68degC Heat-stable Catalase test
Nitrate Reduction
Niacin accumulation
Tween 80 hydrolysis
RGT: Culture media with T2H; (+) No growth (Sensitive, M. bovis); (-) Growth (Resistant, M. tuberculosis)
RGT: Ferric ammonium sulfate and Pyrazinoic acid; (+): Red/Pink band in 4 days (M. intracellulare); (-): Colorless/Gray (M. bovis)
RGT: Iron-uptake test 20% ferric ammonium citrate; (+): Rusty brown colonies in egg-based media (M. smegmatis and M. fortuitum)
(-) No color change (the rest of Mycobacteria)
RGT: Urea broth; (+): Pink/Red in 3 days (M. tuberculosis); (-): No color change (M. gordonae)
RGT: 5% NaCl added to egg-based media; (+) Substantial Growth
(M. fortuitum); (-): No growth(M. gordonae)
T2H Inhibition
Pyrazinamidase test
Iron-uptake test
Urease test
Salt tolerance test
Causes Leprosy (Hansen disease)
__ phase: Multiplication in skin macrophage
__ phase: Multiplication in peripheral nerves
__ form: Tuberculoid leprosy
Disseminated form: Lepromatous leprosy (Facial and nasal deformities, Lepromin skin test negative)
Laboratory methods:
____ appearance under the microscope
NOTCULTIVABLE in regular media (in vitro) but Cultivable in ___ or mice (in vivo). Phenolase test positive; Fite faraco stain
Mycobacterium leprae
Silent
Intermediate
Localized
ciagrette packet/picket fence
foot pads of armadillo
General characteristics: Catalase and Oxidase positive, Gram-negative diplococci, Capnophilic (5-10% CO2).
___ are fastidious/fragile organisms that are temperature-dependent and are inhibited by __ and increased SPS concentrations (0.025% lang dapat).
Hold cultures for up __ in CO2 incubator before reporting as negative/no growth. Other Neisseria spp. are environmental isolates and has low virulence and associated to opportunistic infections.
Gram-Negative Cocci
Neisseria; fatty acid
72 hrs
NEISSERIA
T1 and T2 are virulent strains; Reduced recovery: >30 mins. delay,
use of cotton tip or calcium alginate swab, oil-based lubricant.
MOT: sexual contact or verrtiacl transmission
VF: Pili (T1-T5), Capsule, Endotoxin, Protein II (Opa), Protein II (RMP),
Porin (PorB); can cause Perihepatitis (Fits-High-Curtis)
PCR/NAAT specimens: Endocervical/urethral swab or urine
__-bean or __-bean shaped diplococci inside PMN.
Selective media: ___ agar; Culture media: CAP, __ (3)
AST media: gonoccocus agar (GC agar)
~ 4+ in __; CTA: __ only
Neisseria gonorrhoeae
Kidney; coffee
GC-LECT
Superoxol test (30% H2O2); glucose
NEISSERIA:
MOT: from respiratory droplets, close contact
VF: IgA protease, Proteins like Por and Opa; can cause Waterhouse friderichsen syndrome
Specimen: CSF, Blood, NPS, skin; Culture media: BAP, CAP, TMA
~ Catalase and Oxidase +, Beta-galactosidase and GGT +
CTA: __ and __ only (CTA (+) is yellow, (-) is pink)
Neisseria meningitidis
glucose and maltose
NEISSERIA:
Small and translucent colonies with yellow pigment
CTA: ___?; Lactose fermenter, ONPG (+)
dry, wribkled colonies exhibiting bread crumbs
Grows on BAP and CAP at 25degC; CTA: __
Colonizers of upper respiratory tract and female genitalia.
Transmission through respiratory droplets; __, __ are responsible for beta-lactamase production; ___________________ (intact colonies)
or old “Wagon wheel” appearance, __ , Beta-lactamase +, Butyrate+, Can grow on Nutrient agar
Neisseria lactamica; Lactose, Glucose, Maltose
Neisseria sicca: Sucrose, Glucose, Maltose, Fructose
Moraxella catarrhalis; BRO-1, BRO-2; hockey puck colonies; DNAse +
Rapid ID of M. catarrhalis; (+) result: ___ (M. catarrhalis); (-): No color
change (__)
ID of N. meningitidis: (+) result: __; (-) No color change (M. catarrhalis)
Butyrate/Tributyrin test; Blue/Indigo; N. gonorrhoeae
GGT or GGAP; yellow
Carbohydrate utilization medium: __; Reagent: 1% carbohydrate; Indicator: Phenol Red
Incubate at 35-37degC for 24-72 hours in ambient air.
Positive: __ color. Negative control: ___ CTA tube.
Cystine Trypticase Soy Agar; Yellow; Uninoculated
Oxidase negative, Gram-Negative Bacilli: Enterobacterales
- Gram negative __ bacilli (not curved)
- Opportunistic pathogens except for Overt/True Pathogens: E. coli (Diarrheagenic), Salmonella, Shigella, Plesiomonas, and Yersinia
- All are motile except ___ (mnemonic) ~ though who is motile at room temp 25degC?
- ALL ARE OXIDASE (-) except for ___
- All are catalase + Nitrate reduction + Glucose fermenter
long slender straight
SKY (Shigella, Klebsiella, Yersinia); Yersinia
Plesiomonas
Rapid Lactose fermenters? mnemonic
Late Lactose Fermenter?
Non-Lactose Fermenter?
EcEK: Escherichia coli, Enterobacter, Klebsiella
SeCSea: Serratia, Citrobacter, Salmonella enterica subsp. aizonae; (
ShYPMMS: Salmonella, Shigella, Yersinia, Hafnia, PMP group
Tests for Gram-Negative (Enterobacterales):
Aid in ID of Gram-negatives along with Gram stain; RGT: Longwave UV light; (+) Blue fluorescence (E. coli); (-) No fluorescence (S. aureus)
Differentiates Enterobacterales from other GN bacilli; (+) Dark purple/Deep Blue (P. aeruginosa); (-) No color change (E. coli)
For Enterobacterales members (all +); (+) RED after addition of
indicator. (E. coli); (-) Colorless or Change of color after zinc dust (Acinetobacter baumanii)
Further Enterobacteria test; RGT: Solution A: Sulfanilic acid and Solution B: a-naphthylamine (both reductions); (+) Colorless, gas production (P. mirabilis); (-) Red, no gas (Acinetobacter baumanii)
Tests bacteria for their oxidative or fermentative ability; RGT: Phenol Red; (+) Yellow (acids) Oxidizer: P. aeruginosa, Fermenter: E. coli; (-) none
Gram Sure
Oxidase test (Kovac’s method)
Nitrate Reduction
Nitrite Reduction
Oxidation and Fermentation (CDC Method)
Kauffman-White classification; Serological identification of Enterobacterales spp.:
__: Somatic antigen, Cell wall, Heat-stable, E. coli and Shigella
serotyping
__: Capsular antigen, Capsule, heat-labile; Detection of __ Antigen of E. coli and capsular __ antigen (Virulence) of S. typhi
__: Flagellar antigen, Flagella; Heat-labile; Heat-labile
O antigen
K antigen; K1; Vi
H antigen
Tests for Gram-Negative (Enterobacterales):
For decarboxylase - producing Enterobacteria; RGT: Bromocresol purple and Cresol red;
Lysine →
Ornithine →
Arginine →
Purple:
Lysine – K. pneumoniae
Ornithine – K. aerogenes
Arginine – P. aeruginosa
Yellow:
Lysine – C. freundii
Ornithine – P. vulgaris
Arginine – E. coli
Moeller’s Decarboxylase test
Cadaverine; Putrescine; Citrulline (LOA:CPC)
Tests for Gram-Negative (Enterobacterales):
For Enterobacterales; Aerobic Slant: __; Anaerobic Butt: __; RGT: Bromocresol Purple and Ferric ammonium citrate (H2S)
Lysine decarboxylase →
Lysine deamination →
Glucose fermentation →
H2S produced →
Lysine decarboxylation: (K/K) - Klebsiella, E. coli, Edwardsiella, Salmonella, Serratia
Lysine deamination and glucose fermentation: (R/A) or R/Y: __ group:
Quality Control:
K/K +H2S: C. freundii
K/A: __
R/A: Proteus mirabilis
LIA
Lysine; Glucose
Purple slant (K)
Red slant (R)
Yellow butt (A)
black precipitate
PMP
E. coli
Tests for Gram-Negative (Enterobacterales):For Enterobacterales; Aerobic Slant: Lactose,(__), Sucrose (__),
Anaerobic Butt: Glucose (__ ); RGT: Phenol Red (acid production) and Ferric ammonium citrate (H2S)
RXN: Ⓐ - Acid/Acid + gas
Ⓐ -
K/A, g/G, H2S+ - Salmonella enterica subsp. enterica serovar Typhimurium
K/A, H2S+ - __
K/A – __
Triple Iron Sugar
10%; 10%; 1%
E. coli
P. mirabilis
Shigella flexneri
Tests for Gram-Negative (Enterobacterales):
A: Differentiate enteric bacteria from Coryneforms; B: Differentiate
Enterococci from Streptococci
RGT: (A) Andrade’s indicator & Durham tube; (B) Bromocresol purple.
A: (+) Pink With gas (E. coli), Without gas (S. flexneri); (-) Clear or straw, no gas (Coryneforms)
B: (+) Yellow with gas (E. coli); (-) Pink, no gas (Moraxella osloensis)
Positive for Moraxella lacunata, Serratia orodifera, Proteus spp.,
Pseudomonas; RGT: Refrigeration; (+) Partial (top layer) or total
liquefaction of tube at 4degC (Bacillus subtilis); (-) Complete solidification of tube at 4oC (E. coli)
Distinguishes late lactose fermenter from non-lactose fermenters; (+) Yellow (Shigella sonnei); (-) Colorless (Salmonella enterica serovar Typhimurium)
Fermentation media
Gelatin Hydrolysis
ONPG test (o-Nitrophenyl-β-D-Galactopyranoside)
Tests for Gram-Negative (Enterobacterales):
For IMViC reactions; positive for PMP group, E. coli, Klebsiella oxytoca
Plesiomonas, Edwardsiella, Citrobacter koseri (PEKPEC); RGT: Kovac’s reagent (dimethylamine-benzaldehyde & HCl), Ehrlich’s reagent (__ and __ are added)
(+) Pink/Wine color RING after addition of indicator; Colorless/Yellow
Positive Red: Citrobacter, E. coli, PMP, Salmonella, Shigella (CEPSS); RGT: Methyl Red; (+) RED (E.coli); (-) YELLOW (K. aerogenes)
(+) Klebsiella, Enterobacter, Serratia, Hafnia (KESH); RGT: Solution A: ɑ-naphthol, Solution B: KOH; (+) RED (K. aerogenes); (-) YELLOW
(E.coli)
For IMViC reactions; MED: Simmons citrate agar slant; RGT: Bromothymol blue; (+) Growth; Blue (K. aerogenes); (-) Small/No growth; Green (E. coli)
Differentiate E. coli from Shigella; Ability to use the Sodium
acetate as sole source of carbon; RGT: Bromothymol blue; (+) Growth; Blue (E. coli); (-) Small/No growth; Green (S. sonnei)
Spot Indole
Ehrlich’s reagent
Methyl Red
Voges-Proskauer (Barritt’s method)
Citrate utilization
Acetate Utilization
Tests for Gram-Negative (Enterobacterales):
Presumptive ID of Proteus spp. and other enterobacteria; RGT: Phenol red;
(+) Magenta/Red (P. vulgaris) Other positive organisms: (Klebsiella, PMP group, Nocardia, Rhodococcus, Helicobacter, Ureaplasma, Cryptococcus)
(-) Light Orange/Yellow (E. coli)
Detects motile organisms; Motile bacteria produces diffuse zone of growth; (+) Spreading (P. vulgaris); Colonies remain on site of
stab (K. pneumoniae)
Detection of EHEC/E.coli O157:H7 (no fluorescence); RGT: 366 nm Ultraviolet light; (+) Electric Blue fluorescence All E. coli except O157:H7; (-) No fluorescence (K. pneumoniae) and E. coli O155:H7
Urease (Christensen’s method)
Motility test
MUG test
Tests for Gram-Negative (Enterobacterales):
Detects PMP/PPM group (Positive); RGT: 10% FeCl3; (+) GREEN
(P. mirabilis); (-) Yellow (E. coli)
Rapid detection of Vibrio spp.; __ lyses DNA of VIBRIO only, forming a string; (+) String-like between loop and slide (Vibrio spp.); (-) No string formation
PAD test
String test; 0.5% Sodium deoxycholate
1 cause of community-acquired and hospital-acquired UTI, most common gram-negative healthcare-associated infections, and gram negative sepsis
#2 cause of neonatal meningitis (k1 antigen + E. coli)
Escherichia coli
Pili heat stable (ST); heat labile toxin; Cholera-like toxin (water and electrolyte secretion)
DSE: #1 in Traveler’s diarrhea, Childhood diarrhea, Montezuma’s revenge
Profuse Watery Diarrhea (Cholera-like diarrhea)
Strains: E. coli O4, O8, O25
Oversecretion of cyclic adenosine monoPO4 (cAMP)
ETEC or Enterotoxigenic E. coli
Pathogenicity island virulence factors: Bundle of pili, intimin, toxins:
loss of microvilli
DSE: Infantile diarrhea, Mucoidal diarrhea, Can be in __
Strains: E. coli O55, O111, O114
Seen in infants in low-income nations
EPEC or Enteropathogenic E. coli
Infant formula
Invades large intestine in a manner identical to Shigella. Invasin toxin, Large plasmid, (Sereny test +)
DSE: Dysentery (necrosis, inflammation, and ulceration of large
intestine) Shigella-like manifestations
Strains: E. coli O124, O143, O164
Seen in young children in areas with poor-sanitation
EIEC or Enteroinvasive E. coli
Shiga-like toxin Verotoxin/Verocytotoxin
DSE: Bloody diarrhea, H.U.S. (hemolytic uremic syndrome),
Hemorrhagic colitis
Strains: E. coli O157:H7, O26
Undercooked hamburger, unpasteurized dairy products, apple cider,
bean sprouts, spinach, cookie dough
EHEC / VTEC / STEC or Enterohemorrhagic E. coli
__ – clumped/aggregates in intestine through pili; watery diarrhea
__ – diffuse patterns on HeLa or Hep2 cells, causes diarrhea mostly in children
__ - #1 cause uncomplicated UTI
__ – K1 Antigen positive
Enteroaggregative E. coli (EAEC)
Diffusely-adherent E. coli (DAEC)
Uropathogenic E. coli (UPEC)
Meningitis/sepsis-associated E.coli (MNEC)
Ingestion of contaminated poultry products (milk, eggs, dairy)
All Salmonella are __ except Salmonella serotype Gallinarum and Typhi
All Salmonella produce H2S except __. Salmonella serotype Typhi and Paratyphi persists in __ (gall stones). Cholecystectomy – Tx for chronic carriers
Salmonella sampling:
1st week: __
2nd week: __
3rd week: __
Salmonella spp.
motile
Salmonella serotype Paratyphi A; gall bladder
blood
stool
urine
Salmonella:
__ (Typhoid fever) - Contaminated food from food handlers that are carriers (Typhoid Mary)
Long term carrier: __
Commonly caused by S. enterica subsp. enterica serotype Typhi
Malaise, anorexia, myalgia, severe frontal headache
Complications: __
Hallmark: __ during second week of fever
__ (Food poisoning)
Commonly caused by S. enterica subsp. enterica
Salmonella Typhimurium outbreak (contaminated __ cracker and cereals); Nausea, vomiting, fever, chills, watery diarrhea, abdominal pain
Enteric fever
gallbladder
Necrotizing cholecystitis
Rose spots
Acute Gastroenteritis; peanut butter
Biochemically inert bacteria; Closely related to E. coli
Infections caused by contaminations from Four F’s (?)
Shigella that produces __ weakens agglutination for serotyping. Suspend isolate in saline and heat in waterbath for 15-30 mins at __. Cool then retype with antisera.
Shigella spp.
Flies, Fingers, Food, Fecal
capsule; 100degC
SHIGELLA:
Group A; Most virulent (Shiga toxin); DSE: Bacillary dysentery
NEGATIVE for ONPG, Ornithine Decarboxylase and Mannitol fermentation
Shigella dysenteriae
SHIGELLA :
Group B; Pathogenic, common isolate; DSE: GAY BOWEL SYNDROME!!!; NEGATIVE for ONPG & Ornithine Decarboxylase, mannitol ferm +
Shigella flexneri
SHIGELLA:
Group C; Pathogenic, less incidence; DSE: Dysentery; NEGATIVE for ONPG & Ornithine Decarboxylase, mannitol ferm +
Shigella boydii
Clinically Relevant Enterobacterales spp./Other Pathogenic Enterobacteria:
Non-motile, Urease negative; Class A bioterrorism agent; Vector: Oriental rat flea, Reservoir: Rats; DSE: Bubonic/Glandular plaque & Pulmonary plaque:
Optimum growth temperature: __degC; Bipolar staining/__; __ pattern in broth
Motile at 25degC or RT; Urease positive, Ornithine and Sucrose positive; MOT: thru
undercooked pork, pork intestine, vacuum-packed meat and chicken, dairy & pets; DSE: Waterborne-Enterocolitis & Appendicitis-like syndrome; Cold enrichment
Bull’s eye colonies in __ Peritrichous flagella; in BB: __ contamination
Motile at Room Temp; Urease positive, Ornithine and Sucrose negative; Agent of rodents, Reservoir: Birds; __ colonies with transparent borders in CIN
Pathogenic; Ingestion of contaminated seafood, oyster, shrimps, water; MOT: Entry on Skin cuts; The ONLY oxidase POSITIVE!!! member of Enterobacteriaceae
IMViC: ___ TSI: __
Yersinia pestis; 25-30degC; Safety-pin appearance; Stalactite
Yersinia enterocolitica subsp. enterocolitica; CIN; Packed RBC
Yersinia pseudotuberculosis; Red pin-like
Plesiomonas shigelloides; +,+,-,-; K/A H2S(-)
SHIGELLA:
Group D; Self-limiting, most common isolate; DSE: Dysentery; POSTIVE for ONPG & Ornithine Decarboxylase and mannitol ferm
Shigella sonnei
Other Non-Pathogenic/Opportunistic Enterobacteria:
Food and fish/water contamination; LDC positive, Non-lactose fermenter, Same IMViC to E. coli: ___; TSI: __
__ (red) pigment at 25degC; Bacteremia outbreaks in nurseries, cardiac surgery,
and burn units.
Swarming; Antigens of __ - OX19, OX2; Antigen of __ - OXK; DSE: Acute glomerulonephritis, Urinary tract infection (UTI), Kidney stone formers
~ PAD positive, Urease positive, LDA positive: (5)
Edwardsiella tarda; (+,+,-,-); K/A g+ H2S(+)
Serratia marcescens; Prodigiosin
Proteus; Proteus vulgaris; Proteus mirabilis
~ Proteus, Morganella morganii, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii
Currant jelly sputum, Capsule (Mucoid), Some are Carbapenemase producer; Large pink mucoidal colonies in MAC, ___ positive, __ positive
Blue safety pin appearance; DSE: Granuloma inguinale or Donovanosis; Donovan bodies in endothelial cells
Can break sialic acid in breast milk and infant formula. Contaminant of powdered
infant formula!!!!!; IMViC: __
Nosocomial outbreaks due to contaminated IV fluids; Negative TSI reactions
but positive glucose fermentation (K/A)
May be mistaken as Salmonella in biochemical reaction (Citrobacter is LF); Differentiate with indole or H2S
Klebsiella pneumoniae, potassium cyanide (KCN), String test
Klebsiella (Calymmabacterium) granulomatis
Cronobacter (Enterobacter) sakazakii; -,-,+,+
Pantoea (Enterobacter) agglomerans
Citrobacter freundii (positive for H2S) & Citrobacter koseri (C. diversus) (positive for indole)
ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS:
A/A g K/K + + – – –
MAC: pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
K/A g K/K – – + + –
MAC: Pink(Late)
SSA: Colorless
XLD: Colorless
HEA: Colorless
A/A G K/A – – + + –
MAC: pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
A/A G K/K – – + + +
MAC: pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
Escherichia coli
Serratia marcescens
Enterobacter cloacae
Klebsiella pneumoniae
ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS:
A/A G K/K + – + + +
MAC: pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
A/A G K/K – – + + –
MAC: pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
A/A g K/A + + – + –
MAC: Pink/Red
SSA: Pink/Red
XLD: Yellow
HEA: orange to salmon pink
A/A H2S g K/A – + – + –
MAC: Pink/Red
SSA: P/R (H2S)
XLD: Y (H2S)
HEA: Y (H2S)
Salmonella enteritidis K/A H2S g K/K – + – + –
MAC: Colorless
SSA: C. (H2S)
XLD: R/C.( H2S)
HEA: G/C.(H2S)
MAC:
SSA:
XLD:
HEA:
Klebsiella oxytoca
Enterobacter aerogenes
Citrobacter diversus
Citrobacter freundii
Salmonella enteritidis
ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS:
K/A H2S v R/A + + – + +
MAC: Colorless
SSA: Colorless (H2S)
XLD: R/C (H2S)
HEA: G/C (H2S)
K/A H2S g R/A – + – + +
MAC: Colorless
SSA: Colorless (H2S)
XLD: R/C (H2S)
HEA: G/C (H2S)
K/A H2S K/K – + – – –
MAC: Colorless
SSA: C. (H2S)
XLD: R/C.( H2S)
HEA: G/C.( H2S)
K/A K/A – + – – –
MAC: Colorless
SSA: Colorless
XLD: Red/C
HEA: Green/C
K/A K/A – -v – – –
MAC: Colorless
SSA: Colorless
XLD: Red/C
HEA: Green/C
Proteus vulgaris
Proteus mirabilis
Salmonella serotype Typhi
Shigella dysenteriae & Shigella sonnei
Shigella flexneri & Shigella boydii
K/A H2S g K/K + + – – –
MAC: Colorless
SSA: C. (H2S)
XLD: R/C.( H2S)
HEA: G/C.( H2S)
K/A R/A + + – + +
MAC: Colorless
SSA: Colorless
XLD: Red Or Colorless
HEA:Red Or Colorless
K/A R/A + + – + –
MAC: Colorless
SSA: Colorless
XLD: Red Or Colorless
HEA:Red Or Colorless
K/A g R/A + + – – +
MAC: Colorless
SSA: Colorless
XLD: Red Or Colorless
HEA:Red Or Colorless
K/A K/A v + – – +
MAC: Colorless
SSA: Colorless
XLD: Red Or Colorless
HEA:Red Or Colorless
Edwardsiella tarda
Providencia rettgeri
Providencia alcalifaciens
Morganella morganii
Yersinia enterocolitica
Curved, Oxidase positive, Gram-Negative Bacilli:
General characteristics: Agents of gastroenteritis, Catalase Positive, Oxidase Positive
Genus: (4)
__ – Gram negative Curved/Comma-Shaped Bacilli, Catalase, Oxidase, Indole, Glucose Positive, Motile via __, Facultative Anaerobe; Found in ___; __ except V. cholera and V. mimicus; Infections: Cholera, Septicemia, Wound infections
Vibrio, Campylobacter, Arcobacter, Helicobacter.
Vibrio spp.; Polar flagella; Salt/Brackish/Marine water; Halophilic
Pandemic strain: VC-O1
Biotypes: __ - VP +, B-hemolytic, agglutinates chicken RBC, polymyxin B resistant; Classical
Serotypes: Ogawa, Inaba, Hikojima
VF: Choleragen, Cholera toxin (CT), Zonula occludens toxin (Zot), Accessory cholera toxin (Ace), Flagella, Mucinase, Toxin Coregulated Pili (TCP); DSE: Cholera
HALLMARK: __; Transport media: Cary-Blair NO TO GLYCEROL!!
Enrichment media: pH 8.4; agar used?
Culture media: pH 8.6; agar used?
String test positive, Vibriostatic test (0129-impregnated disk) sensitive
Vibrio cholerae; El Tor; rice watery stool
APW; Alkaline peptone water; TCBS
V. cholerae (Cholera; Rice water stool)
V. alginolyticus (Otitis, wound infection, gastroenteritis)\
V. furnissii
V. fluvialis
V. cincinnatiensis
V. metschnikovii
Sucrose Fermenter ; Yellow in TCBS
Non-Sucrose Fermenter ; Green in TCBS
causes Summer diarrhea, B-hemolysis in Wagatsuma agar
causes Septicemia, wound infection, Lactose +
V. mimicus
V. parahemolyticus
V. vulnificus
Aquatic environments, Freshwater, polluted, chlorinated, brackish water, occasionally marine water
DSE: Human: Gastroenteritis, wound infection, bacteremia
Reptile: ___
Gram negative, straight bacilli with rounded ends, Oxidase positive __ colonies on CIN
__-hemolytic on BAP, Do not grow on TCBS., __ , grows with 0% NaCl
Aeromonas spp.
Red-leg syndrome
Bull’s eye, Beta
NOT halophilic
Gram-negative Curved/Seagull wing-shaped bacilli
Catalase, Oxidase, _______ Positive; Microaerophilic. Grows on 42degC (Hot enrichment method); DSE: Associated with gastroenteritis, reactive arthritis (Reiter syndrome) and ___
Improved staining: 0.1% basic fuchsin
Culture media: Skirrow, CAMPY-CVA, Cefoperazone agar, Charcoal-based selective medium (CSM)
DSE: Causes gastroenteritis and septicemia; __ method enhance recovery of Campylobacter and __ in stool samples using 0.45 to 0.65 um pore size polycarbonate or cellulose acetate filters; Plate is __ for 1 hour at 37oC.
~ __ gram negative bacilli that are Oxidase positive, __ Catalase positive, & Urease NEGATIVE
Campylobacter spp.
indoxyl acetate; Guillain-Barre syndrome
Arcobacter spp.; Filtration; Arcobacter; upright
Curved; weak
DSE: Causes Peptic Ulcer & Peptic Cancer
Genetic Virulence:
- __ produces exotoxin that creates vacuoles and loosens tight junctions
- __ is a part of Pathogenicity islands causing peptic carcinoma
- __ binds Leb antigens in gastric epithelial cells
- __ causes peptic ulcer
Other Helicobacter: H. rappini, H. cinaedi, H. fenneliae – isolated from bacteremic homosexual men with HIV;
~ Catalase, Oxidase, Motility, Urease Positive!!! (Urea Breath Test)
Microaerophilic, Curved Gram negative bacilli; Tissue biopsy transport media: Brucella broth with glycerol, Stuart’s transport medium, Portagerm pylori media
Stain: ___ (3)
Helicobacter pylori
VacA
CagA
BabA
IceA
0.1% basic Fuchsin, Warthin-starry (silver), Giemsa
Oxidase positive, Gram-Negative Bacilli
General characteristics: Aerobic, Non-spore forming, & Motile with __ (except Burkholderia mallei)
Genus: Pseudomonas, Burkholderia, Cupriavidus, Ralstonia,
Agent of Nosocomial infection, Infection of wound (Blue pus), Burn
and Diabetic patients, Cystic Fibrosis and COPD patients, Dermatitis in __ and __, Ecthyma gangrenosum – black and necrotic skin papules, ___ AKA Malignant Otitis externa, __ solution contamination
~Catalase, Oxidase +, Acetamide +, Cetrimide growth +, Growth at __
__ odor (2-Acetaminophenone); Virulence factors: __ (kill host cell through protein synthesis inhibition), Elastase, Phospholipase C, Pili,
___ (disrupt cytoskeleton)
Pigments: __, (Pyorubin, Pyoverdin, Pyomelanin)
Biofilm producer (Alginate) – inhibitor of phagocytosis
___ – detects bacterial products in environment
polar flagella
Pseudomonas aeruginosa; hot tubs and Jacuzzi; Swimmer’s ear infection; Contact lens
42degC, Grape-like; Exotoxin A; Exoenzyme S and T
Pyocyanin
Quorum sensing
Infection on patients with cystic fibrosis: __
Plant pathogen that is isolated from on patients with cystic fibrosis and chronic granulomatous disease (CGD): ___
Burkholderia mallei: Causes severe infection in horses (_____ disease)
Burkholderia pseudomallei or __; Melioidosis/Glander’s-like disease AKA the _____ or Great Mimicker; Sample: __ (3) ; Dry, Violet-purple, Wrinkled colony on __
Common in environment; Brown/yellow Adherent Wrinkled colonies
Nosocomial/catheter-related infections. ______. Beta-lactamase +.
LDC, DNase and Motility positive. ___ in BAP. __ odor
Burkholderia cepacia
Glander’s disease
(Whitmore’s bacillus); Vietnamese time bomb; Throat swab, Rectal swab, Sputum; Ashdown’s agar
Pseudomonas stutzeri
Stenotrophomonas maltophilia; oxidase negative; Lavender green; Ammonia
Drug resistant. Nosocomial infection. An ESKAPE pathogen. ________
Catalase + __ in MAC. __ of carbohydrates (A. lwofii is not an oxidizer) __ coccobacilli, resist decolorization
Causes Conjunctivitis; Gelatinase +. Liquefy/Digests ___
slant.
Causes neonatal meningitis; Old genus: Flavobacterium, Chryseobacterium; Produce __ pigment; Oxidase and DNase +
Associated to chronic granulomatous disease (CGD), sepsis, and necrotizing fasciitis; Violacein pigment. Almond odor/____ odor.
Acinetobacter baumanii; oxidaaaaase negative (dami a); Lavender; Oxidizer; Plump
Moraxella lacunata; Loeffler’s serum
Elizabethkingia meningoseptica; flavin/Yellow.
Chromobacterium violaceum; ammonium cyanide
ESKAPE GROUP – group of highly virulent, antibiotic-resistant, nosocomial infection-associated bacteria.
Small, nonmotile, capnophilic, pleomorphic Gram negative bacilli or coccobacilli; Fastidious/fragile organisms requiring __ and __. Capsule is composed of Sugar-alcohol (polyribitol) phosphate; __ capsular serotype: H. influenzae type a to f
__ – Normal Flora, “Satellite Phenomenon”; Meningitis, Epiglottitis, Cellulitis, Arthritis, Pneumonia; Otitis Media: transmitted through __. Particle agglutination for CSF and urine.
__ - Brazilian purpuric fever
__ – Acute otitis media, sinusitis, bacteremia, culture negative endocarditis
__ – purulent conjunctivitis, pink eye
__ – Chancroid/Soft chancre (genital lesions → papules → painful ulcers), a STD; School of red fish
Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter spp.
Haemophilus spp.; X and V factors; 6
H. influenzae; respiratory droplets
H. influenzae biotype aegypticus
H. parainfluenzae
H. aegypticus
H. ducreyi
HAEMOPHILUS SPP:
group that needs X and V factor but dont produce porphyrin
group that needs V factor and produce porphyrin
group that needs X factor ONLY
group that only produces poprphyrin
H. influenzae, H. aegypticus, H. haemolyticus (1st row)
H. parainfluenzae, H.parahaemolyticus, H. paraphrophilus (PiPhaPaP or p- pop) [2nd row]
H. ducreyi {3rd row}
Aggregatibacter aphrophilus
To differentiate Haemophilus spp.
Media: Heart infusion agar, TSA, NA, Haemophilus medium
Reagent: __ impregnated with ___ factor; Incubate: Overnight, 35oC, ambient air
Positive results:
Growth around XV disk only: 1st row of the table
Growth around XV and V disks only: 2nd row of the table
Growth around XV and X disks only: 3rd row of the table
Negative result:
Growth around XV, X, and V disks: __
X and V factor test
Disk/strip; X, V, & XV
Non-fastidious organisms
- 5 dysgonic small gram negative (cocco) bacilli
- Normal oral flora but occasionally associated with Subacute Bacterial Endocarditis!!!!
- Recovered in blood culture then Subculture in BAP/CAP (Candle jar incubation)
A: seen in Dental plaques, X factor dependent, Nitrate reduction +
A: seen in Periodontitis; Coinfection with Actinomyces; Star shaped centers in BHIA
C: Frequently associated with aortic valve infection; The only Indole positive in AACEK!!!!! False gram positive staining - ___; Rosette (rose petal) formation with stick-like projections under microscope
E: Human bite infection Bleach-like odor, ______
K: Joint and bone infections; osteoarthritis in children Narrow zone of beta hemolysis in BAP
AACEK (HACEK):
Aggregatibacter (Haemophilus) aphrophilus
Aggregatibacter (Actinobacillus) actinomycetemcomitans
Cardiobacterium hominis; pits agar
Eikenella corrodens; pits agar
Kingella kingae
CATALASE, OXIDASE +; BEST Specimen: Nasopharyngeal swab (NPS)
Virulence: Pili; Causes __ cough/100 days fever (Catarrhal, Paroxysmal, Convalescent stage); Prevented with DPT vaccine
Media: Potato Blood glycerol/Bordet Gengou agar, Regan Lowe/Charcoal Cephalexin Blood agar, Casamino broth, Jones Kendrich
Bordetella pertussis; whooping
Bordetella:
- the only oxidase positive!!
- grows on BAP and uerase positive
- positive in Motility, Nitrate reduction, Oxidase, Urease, Growth
B. pertussis
B. parapertussis
B. bronchiseptica
Thionine and Fuchsin inhibition tests is used for:
only specie inhibited by thionine is: __
species inhibited by fuchsin is: __
not inhibited by both is: __
DSE: Animal and Human pathogen ___, Undulant fever, __, ___, Mediterranean fever, Animal abortion; Found in animal placenta/erythritol.
Transmission: Ingestion of Unpasteurized or
contaminated milk/cheese, Inhalation through animal carcasses, needle-stick/skin abrasions
Common lab-acquired infection through aerosols
Brucella spp.
B. abortus
B. suis, B. canis
B. melitensis
Brucellosis; Bangs disease; Gibraltar
disease
GRAM NEGATIVES:
Deerfly fever, Rabbit fever; Require Cysteine & Cystine. __ skin test. Glucose Cysteine Blood agar, Peptone Cysteine agar.
Shipping fever. Bioterrorism. Cat-Bite fever!!! Oxidase, Catalase, Glucose, Indole +;_______ odor
Cat-Scratch disease (#1)!!! Oxidase, Catalase negative. Warthin Starry stain.
causes Cat-scratch disease
Dog-bite infection, Periodontal disease; Gliding motility, Fusiform shaped Gram negative; Sensitive to SPS
Rat-Bite fever/Sodoku!!!; dse Helix shaped. Sensitive to SPS
Oroya fever, Verruga peruana/__, Carrion’s disease!!!; History. Vector: __
Francisella tularensis; Forshay
Pasteurella multocida; mushroom
Bartonella henselae
Afipia felis
Capnocytophaga spp.
Spirillum minor/minus
Bartonella bacilliformis; Peruvian warts; Sandfly
GRAM NEGATIVES:
Haverhill fever; Broth: __ and fluff balls appearance; Agar: __ appearance with dark center and __; Stained with __ for Microscopy
Broadstreet pneumonia AKA __. Pontiac fever (no pneumonia); Faint in Gram stain, use __ or 10 mins. Safranin. From Air conditioning unit, condensers, humidifiers, nebulizers, & cooling towers. DFA to detect antigens in urine, respiratory samples, & tissues.
Normal flora of vagina; Bacterial vaginosis; Nugent scoring system: __; __ –10% KOH __ odor;
Gram-variable or gram-negative coccobacilli and
short bacilli, Catalase neg, Oxidase neg, Inhibited by __; Hippurate positive, __
Primary Atypical Pneumonia or __, Pleuropneumonia-like organism (PPLO),
Stevens-Johnson syndrome; Confirmed using HEMADSORPTION (using guinea pig RBC) from Edward Hayflicks medium. Use SP4 media with GLUCOSE or NYC agar;
Post-abortal, post-partum fever; HIV-associated cervicitis; __, embedded in media. Use SP4 media with __,
NGU, PID, Prostatitis, Infertility, Sexually acquired arthritis, Enhances HIV transmission;
Glucose metabolism Positive, __ metabolism and Urease Negative
Urethritis (NGU), neonatal infection; Use SP4 media with UREA or NYC agar
Streptobacillus moniliformis; Breadcrumbs; Fried egg; lacy edges; Dienes
Legionella pneumophila; Legionnaire’s disease; Fuchsin
Gardnerella vaginalis; Clue cells; Whiff/Sniff test; Fishy-amine odor; SPS; HBT agar
Mycoplasma pneumonia; Walking pneumonia
Mycoplasma hominis; Mulberry/Fried egg colonies; ARGININE
Mycoplasma genitalium; Arginine
Ureaplasma urealyticum
ANAEROBIC BACTERIOLOGY:
__ percent Oxygen requirement – ___ (throat, NPS, rectal, etc.); Holding temperature: ROOM TEMP (20-25oC).
Culture media: PRAS media, Reduced media, Roberston’s Cooked/__ media, Thioglycollate broth, Anaerobic blood agar, AnaeroPak system, Gas Pak Jar system, Brewer jar, McIntosh and Filde’s jar, Boiling
Indicator of Anaerobiosis: ___
ZERO; NO SWAB
Chopped meat
Resazurin and Methylene blue (BLUE)
Indicators of the Prescence of
Anaerobic Bacteria
1. __ odor
2. __ granules
3. __ fluorescence on UV light
4. __ of hemolysis
5. Lack of __
Foul, Sulfur, Brick red, double zone; Superoxide dismutase
Obligate intracellular Nonculturable Bacteria:
Energy parasites/Obligate Intracellular
__ are like gram-negative in a way that they have LPS as well in their cell wall. Antigenic variations is due to __; __ body – Metabolically Inert, Infective form; __ body – Intracellular, Active Replicating form
Chlamydia spp.
Chlamydia; MOMP (Major Outer Membrane Protein); Elementary; Reticulate
ANAEROBES:
Gas gangrene bacillus; VF: Encapsulated, Central to Subterminal spores (Histotoxin)!!!! __-shaped
appearance; Double zone of hemolysis, __ positive on EYA; Nagler test +; __ +; DSE: Gas gangrene, Necrosis, __ disease
Tack-Head bacillus; ____spore, Tetanospasmin
(Neurotoxin); Drumstick/lollipop/Racquet appearance; Swarming on BAP, Lecithinase negative; DSE: Tetanus, Lock jaw Trismus, __, muscular rigidity and __
Caned good bacillus; _____spore (Neurotoxin); With __ spore resembling tennis racket; __ positive on EYA ___ bioassay – Identification of
Botulinum toxin (BoNT); DSE: Foodborne botulism, Botulism, __ syndrome, __ (rag doll)
paralysis; blurred vision, slurred speech, dysphagia
________: gold standard for detection of toxin
Toxin A (TcdA) – __; Toxin B (TcdB) – __; Toxin C and binary toxin; Rods in chains up to __.
With __ spores; __ on CCFA; __ odor on BAP;DSE: Pseudomembranous colitis
C. perfringens; Boxcar; Lecithinase ; Reverse CAMP; Pig-bel
C. tetani; terminal spore; Risus
sardonicus; spasm
C. botulinum; subterminal; Lipase; mouse; Floppy baby; Flaccid
C. difficile; tissue/cell culture; enterotoxin; cytotoxin; 6; oval; Yellow ground glass; Horse manure
Obligate intracellular Nonculturable Bacteria:
#1 Ocular Trachoma (Serovars A, B, C)
#1 Pelvic Inflammatory disease (Serovars D to K)
#1 Lymphogranuloma venereum (Serovars L1, L2, L3)
#1 Non-Gonococcal urethritis
#1 STD in __
TRachoma and Inclusion Conjunctivitis agent
__ syndrome
TESTS: Use __ shafts of swab (Endocervix, Vaginal, Urethral)
Best Media: __
Growth enhancer: __
Best Test: PCR/NAAT
Test Chlamydia antibody:
Microimmunofluorescence
inclusions: __ bodies; Glycogen-containing bodies; Stain: __
Chlamydia trachomatis
U.S.; Fits-Hugh-Curtis
plastic/metal; McCoy Cell Line; Cycloheximide
Halberstadler-Prowazeik; Lugol’s iodine
Obligate intracellular Nonculturable Bacteria:
Psittacosis, Ornithosis, Parrot fever, Pneumonia (__ cough); -from inhalation of bacteria in __
Sample: Sputum, NPS, OPS
Media: ___
Microimmunofluorescence and PCR
inclusions: __ bodies; Non-Glycogen containing
stain: __ and __
Chlamydophila psittaci; Hacking; bird’s
dropping/feces;
Mice or chick embryo
Levinthal-Lillicole
Macchiavello and Giemsa
Obligate intracellular Nonculturable Bacteria:
TWAR strain; Taiwanese Acute Respiratory Syndrome Ass. To asthma and atherosclerosis
Sample: Sputum, NPS, OPS, __
Media: HeLa or Hep-2
Microimmunofluorescence and PCR
stain: Giemsa
Chlamydophila pneumoniae; bronchial wash
TWAR strain of pneumonia
Currant jelly-like sputum
Rust colored sputum; #1 community acquired pneumonia
Primary Atypical Pneumonia (PAP)
Broadstreet pneumonia
Chlamydophila pneumoniae
Klebsiella pneumoniae
Streptococcus pneumoniae
Mycoplasma pneumoniae
Legionella pneumoniae
Cell wall is like a Gram-negative cell wall. Obligate intracellular and non-cultivable in cell-free media; Attachment to host cell - ____
Infection occurs after a bite of an infected __ vector; Test is usually: NAAT (PCR) or Serology (e.g. Weil-Felix)
Rickettsia; Omp A & Omp B; arthropod
Spotted Fever group:
African tick bite fever
Mediterranean/Israeli/Boutonneuse spotted fever; Indian/Kenya tick typhus
Far-Eastern tick-borne rickettsiosis
Flinders Island spotted fever
Japanese spotted fever
Rocky Mountain spotted fever
Mild illness
Rickettsia sibirica North Asia tick typhus
Tick-borne lymphadenopathy
Rickettsia africae
Rickettsia conoriii
Rickettsia heliongjiangesis
Rickettsia honei
Rickettsia japonica
Rickettsia rickettsi
Rickettsia parkeri
Rickettsia parkeri
Rickettsia sibirica
Rickettsia slovaca
vector: Ticks
Typhus group:
Epidemic typhus; vector: Lice
Brill-Zinsser disease (Recrudescent disease); vector: None
Murine typhus; vector: Fleas
Scrub typhus group:
Scrub typhus; vector: Chiggers
Others:
Human MONOCYTIC; vector: ehrlichiosis Ticks (Ambylomma spp., Lone star tick)
Human GRANULOCYTIC naplasmosis; vector: Ticks (Ixodes spp.)
Sennetsu fever; vector: Ticks
Rickettsia prowazekii
Rickettsia typhi
~~
Orientia tsutsugamushi
~~
Ehrlichia chaffeensis
Anaplasma
Neorickettsia sennetsu
Is a gram negative coccobacillus which causes Q (Query) fever!!!! and can be Isolated from animals. Survives extracellularly and can only be grown in __ cells. No rash development.
Phase I (Large-cell variant):
Phase II (Small-cell variant): Acting like a __, extracellular (in the environment)
Zoonotic infection with cattle, sheep, & goats
Biosafety level ____
Tests: Shell vial culture using Human lung fibroblast (1 day)
Serology -
IFA -
PCR -
Coxiella burnetti; lung
spore
3
most common test
reference method
more sensitive test than sero
General characteristics: Long, slender, __ curved __ organism
Examined under __ microscope
__ – platelet-like structure in the cell wall in which fibrils are attached
Axial fibrils/Axial filaments/Endoflagella – flagella-like organelles wrapped around the bacterial cell wall
Genus and their morphology:
- __ (Tight coils)
- __ (Thicker, Loose coil)
- __ (Thicker, Loose coil, Hooked ends)
- __ (Comma-shaped/Helical, Tapered ends with 4 flagella on each end)
Spirochetes; helically; microaerophilic; dark-field; Insertion disks
Treponema
Borrelia
Leptospira
Brachyspira (spiral)
Hutchinson’s triad: ______, _____, Notched peg-shaped teeth; Bone malformations: Bulldog maxilla, Saber shin (tibia); Blood or CSF X-ray, Immunologic test
Congenital syphilis; deafness, blindess
SYPHILIS STAGES:
Primary syphilis: __ Chancre in genitals; SPX: Lesion Swab/Aspirate; TEST: Dark-Field microscope, Culture
Secondary syphilis: __ rashes in palms and
soles; SPX: Lesion aspirate; TEST: Sero
Latent syphilis: __ phase; No signs/symptoms;SPX: Serum; TEST: Serology
Tertiary syphilis: Tissue-destruction; Granuloma-like lesions (__). Cardiac and CNS problems, ___
SPX: Lesion. Blood. CSF. Serum. Tissues; TEST: Serology, PCR – Neurosyphilis in AIDS patients
Hunterian chancre/Hard
Condylomata lata
Subclinical
Gummas; Neurosyphilis
Relapsing fever: ____ - stained Blood smear
Blood and CSF (1st week); Urine (2nd week)
Culture: Fletcher, Noguchi, EMJH media
Gold standard-__
Blood (Acridine orange or Giemsa), biopsy (Warthin-Starry stain), CSF, or Urine; Culture: Chick embryo, Barbour-Stoenner-Kelly media; Confirmatory – __
Reaction from teteracycline treatment of spirochetes
Giemsa
Leptospirosis; Microscopic agglutination (MIT)
Lyme’s disease; Western blot
Jarisch Herxheimer reaction
Species of Spirochetes:
Syphilis
Yaws or Fambresia tropica
Bejel or Endemic Non-venereal syphilis
Pinta or Carate
Gingivitis, Periodontitis
Louse-borne/Epidemic/European relapsing fever
Tick-borne/Endemic/American relapsing fever
Lyme’s disease!!!!
Leptospirosis / Infectious Jaundice / Weil’s disease!!!
Treponema pallidum subsp. pallidum
Treponema pallidum subsp. pertenue
Treponema pallidum subsp. endemicum
Treponema carateum
Treponema denticola and Treponema socranski
Borrelia recurrentis
Borrelia hermsii, B. turicatae, B. duttoni, B. parkeri
Borrelia burgdorferi
Leptospira interrogans
Stages of Lyme’s disease:
Erythema chronicum migrans, Fever, malaise, joint pain
Second stage Neurologic disorders (nerve palsy), meningitis
Acrodermatitis chronica atrophicans (recurring chronic arthritis) & Demyelination symptoms of Alzheimer’s disease and Multiple sclerosis
First stage
Second stage
Third stage
QUALITY CONTROL IN MICROBIOLOGY:
Laboratory goals of __:
* Improving the accuracy of patient identification
* Improving the effectiveness of communication among caregivers
* Reducing the risk of healthcare-associated infections.
__: is associated with the internal activities that ensure diagnostic test accuracy. It is defined as
the measures designed to ensure the medical reliability of laboratory data.
__: is associated with the external activities that ensure positive patient outcomes.
__: concentrates on eliminating redundant motion, recognizing waste, and identifying what creates value from the client’s perspective
__: is a concept introduced by Motorola in 1986 that reduces defects by minimizing variation in
processes through metrics measurement. Methodology involves DMAIC
__: Defined as the accuracy, reliability, and timeliness of reported test results.
The Joint Commission (TJC)
Quality control
Quality Assurance
LEAN
Six Sigma
Laboratory quality
International Organization for Standardization (ISO):
__ - Medical laboratories requirements for quality and competence.
__ - General requirements for the competence of testing and calibration laboratories.
__ - General requirements for the competence of providers of proficiency testing schemes
ISO 15189:2007
ISO/IEC 17025:2005
ISO/ IEC 17043:2010
Classification of errors in AST reporting:
* __: adequate interpretation of the susceptibility test result.
* __: Microorganism shows intermediate sensitivity to a given antimicrobial but is reported as susceptible or resistant; microorganism is susceptible or resistant but reported as showing intermediate resistance.
* __: classification as resistant of a susceptible strain (false resistance).
* __: classification as susceptible of a resistant strain (false susceptibility).
In-use/working culture: 1 vial per week is thawed and _______
No errors in interpretation
Minor error (Mi)
Major error (Ma)
Very major error (Vma)
subcultured twice
Lab-related Government Agencies and Function:
- Oversight of public health and safety including the laboratory
- Regulation and Approval of in-vitro reagent kits
- Calibration of thermometer, weights and measures; International system of Units
- Culture media quality control; Format of ____
Standardization of laboratory practices - Control organisms used for AST (20 days testing)
- Licensing of laboratories
- Test kit or method validation, Proficiency testing; Bioterrorism investigation and confirmation along with CDC
Provides Research, education, and training in the field of occupational safety and health; Certification of respirator masks (e.g. N95)
CDC (Centers for Disease Control and Prevention)
FDA (Food and Drug Administration)
NIST (National Institute of Standards and Technology)
CLSI (Clinical and Laboratory Standards
Institute); SOP manuals
ATCC (American Type Culture Collection)
DOH (Department of Health)
RITM (Research Institute for Tropical
Medicine)
NIOSH (National Institute of Occupational
Safety and Health)
RITM NEQAS Frequency:
Bacteriology
Parasitology
Mycobacteriology Microscopy and Culture
- Transfusion Transmissible infections (HIV, HBV, HCV, Syphilis, Malaria): __
SARS-CoV-2 Molecular
Once a year - all except TTIs
Twice a year - TTIs only
Record/Specimen type Retention:
Urine: __
Microbiology stained slides: __
Body fluid and Smears: __
Test Requisitions: __
Patient records: __
Culture media performance and sterility records: __
QC records: __
Retired Laboratory procedures: __
Maintenance of records with billing concerns: __
QC record of instrument: __
urine: 24 hrs
slides, body fluid and smears: 7 days
test req-retired: 2 yrs
maintenance: 10 years
qc record: As long as instrument is being used
DIAGNOSTIC WATER BACTERIOLOGY
___– index of fecal contamination in a drinking water.
__ – indicator organisms of contamination in water.
__ parameters to be tested (DOH): E. coli/coliform, Arsenic, Cadmium, Lead, Nitrate, Color, Turbidity, pH, total dissolved solids, and disinfectant residue.
Bacteriologic analysis of water: (1) Total plate count; (2) Presence/absence of coliform; (3) MPN count
Methods of Microbiologic Water analysis: Multiple tube fermentation (MPN count), Membrane filtration, Defined substrate, Presence/absence of coliform, Pour plate method, Chromogenic substance
E. coli
coliforms
10 Mandatory water
_DIAGNOSTIC WATER BACTERIOLOGY:
_____ – presence of residual chlorine indicates there’s sufficient amount of chlorine to inactivate bacteria/viruses and that water is protected from recontamination!!!! during storage. Residual chlorine should be __ 30mins. after addition of NaOCl and __ 24 hours after addition of NaOCl in water.
______ – neutralizes free chlorine in swimming pool water.
NOTES: Incubation of broth/cultures are mostly 24-48 hours at 35oC (others were up to 44.5oC to detect thermotolerant E. coli). Positive results include _____ in broths and __ colonies in plated media. Indicators commonly used are __ (for acid production, yellow) and __ (for gas detection, bubble).
Chlorine residual testing; 2 mg/L; 0.2 mg/L
sodium thiosulfate
acid and gas production; characteristic coliform!!!!; phenol red; durham tube
- Quantitative (Reported in Most Probable Number → the number of coliform resulting from multiple-portion decimal dilution plantings/ determines number of coliform /checks for the potability of water)
-Gold standard is __ of test tubes. MPN is __ from a standard chart based on no. of tubes with positive result.
__: >1.1 MPN/100 mL
__: <1.1 MPN/100 mL
Multiple Tube Fermentation test (MTFT)
5 set; calculated
Positive
Negative
Stage 1: __
> Lauryl Tryptose broth / Lactose broth + water sample (Incubate at 35oC for 24-48hrs) → Acid and Gas production (+)
Stage 2: __
> To confirm __, use __ medium, otherwise for __ use __.
> Brilliant green lactose bile broth (BGLBB) or E. coli (EC) broth + inoculum from stage1 → gas
production+!!! (NRL manual)
> EMB / Endo agar + inoculum (from stage1) (Incubate at 35oC for 24-48hrs) → colonies (Observe green metallic sheen!!!) (Conventional methods in U.S.)
Stage 3: __
> Single strength Lauryl Tryptose broth + inoculum → Gas production (+) and LES Endo agar or MacConkey agar look for gram negative bacilli, then perform traditional IMViC for differentiation of coliforms.
(DOH, NRL manual)
> Lactose broth + inoculum (from stage 2) Incubate at 35oC for 24-48hrs) → look for acid and gas production (Conventional methods in U.S.)
Presumptive test
Confirmatory test
TOTAL COLIFORM; BGLBB
FECAL COLIFORM; EC broth
Completed test
___ (Formerly: Standard Plate count) Negative: <500 CFU/mL (Dialysis water <200 CFU/mL)
-estimates the number of live heterotrophic bacteria in water; measures changes during water treatment and in swimming pool
-Indirect indication of pathogen removal from water treatment infacilities.
METHODS: Pour plate method, Spread plate method, and Membrane filter method.
CULTURE MEDIA: R2A, NWRI agar, PCA
__ – detects coliforms from the environment, food, and beverage (Incubation is 35degC for
24 hours; characteristic color forms in the presence of coliform bacteria).
Heterotrophic Plate count
Millipore water sampler
MILK BACTERIOLOGY:
Pathogenic: Coxiella burnetti, Campylobacter, Brucella, Bacillus anthracis, Streptobacillus, Salmonella, Y. enterocolitica, Listeria, Vibrio, Mycobacterium bovis, and E.coli (EHEC).
___ – should be NEGATIVE, a positive result indicates improper pasteurization.
Analysis of Milk quality:
Milk is serially diluted and plated in culture media for colony counting. The satisfactory result should be comparable to __.
Methods: __ (WBC count for infection), Agar plate count, Lab pasteurized count, __ (fecal coliform).
Colony Counters:
1. Quebec Darkfield colony counter – ___ background, Manual (Wolffheugel grids) or Automatic counter
2. SC6 Colony counter – __ background, Pressure-sensitive counting, connected to computer/printer
3. __ – Manual counting of colonies in a plate. Traditional method like hemocytometer in Hematology.
__ – dye is reduced (decolorized) by oxygen consuming bacteria; the speed of reduction is directly proportional to the number of bacteria present.
Phosphatase (ALP) test
Grade A milk
Somatic cell count; Coliform count
dark
Light or Dark
Tally counter
Methylene blue reduction test
