Bacteriology Flashcards
Belong to the Protista kingdom
Include some eukaryotes, prokaryotes, viruses, viroids, prions
Classified according to structure, chemical composition, and biosynthetic and genetic organization
Microorganisms
Organ of locomotion for prokaryotes
Flagella
Organ of locomotion for eukaryotes
Flagella and pseudopodia
Cell wall less bacteria
Mycoplasma
Ureaplasma
Fungal cell membrane
Ergosterol
Provides shape to bacteria
Cell wall
In bacteria, energy production happen in
Cell membrane
Not cells, not visible to light microscope
Viruses
Bacterial viruses
Bacteriophages or phages
Single-stranded, covalently closed, circular RNA molecules that exists as base-paired, rodlike structures
Cause plant diseases
Viroids
RNA of the hepatitis D virus is ____
Viroid-like
Abnormal proteins
Prions
Infectious particles associated with subacute progressive, degenerative diseases of the CNS (Creutzfeldt-Jacob Disease)
Prions
Alter conformations of a normal cellular protein that autocatalytically form more copies of itself.
Prions
Spiral Sjape
Spirochetes
Borrelia
Leptospira
Treponema
Straight rod or with single rigid curve
Vibrio
Rigid helical rod
Spirillium
Flesuous helical rod
Spirochete
Vary in size and shape
Pleomorphic
ex: Bacterioides, Mycoplasma, Ureaplasma
Example of antibiotic that affect cell wall biosynthesis and may alter a bacteria’s shape
Penicillin
Coffee-bean shaped diplococci
Neisseria
Lancet-shaped diplococci
S. pneumoniae
Palisades
Corynebacterium
Smallest pathogenic bacillus
Haemophilus
Largest pathogenic bacillus
Bacillus anthracis
Mucilaginous envelop that surrounds some bacteria (if present, considered as virulence factor)
Capsule
Structure surrounding a bacterial cell and is external to the cell wall
Polysaccharide
Bacteria without polysaccharide, instead possess poly-D-glutamic acid capsule
Bacillus anthracis
Property of capsule that may increase virulence by preventing phagocytosis
Immunoevasion
Basis of serotying by Quelling reaction: specific capsular swelling with type-specific antiserum
Antigenic
Encapsulated organisms
“Some Nasty Killers Have Some Capsule Protection”
Streptococcus pneumoniae
Neisseria meningitidis
Klebsiella pneumoniae
Haemophilus influenzae
Salmonella typhi
Cryptococcus neoformans
Pseudomonas aeruginosa
Special staining methods
Hiss stain
India ink stain
Loosely arranged extracellular material that surrounds bacterial cells
Slime layer
Found in biofilms
Slime layer
Used by some bacteria to adhere to surfaces like catheters (S. epidermidis)
Biofilm
Organized layer
Capsules
Also known as the peptidoglycan later or murein layer
Cell wall
Functions of _____
Protects bacteria against osmotic pressure
Gives shape to the bacteria
Confer the gram reaction of the bacteria
Usual target of anti-microbial drugs (penicillins, cephalosporins)
Cell wall
Thicker, teichoic acid
Gram-positive cell wall
Composed of a very thick protective peptidoglycan (murein) layer
Gram-positive cell wall
G+ cell wall consists of glycan chains of alternating ___ and ___
NAG and NAM
Negatively charged and contributes to the negativity of the cell wall
Teichoic acid
It may also bind and regulate the movement of cations into and out of the cell
Teichoic acid
Have an outer membrane
Gram (-) cell wall
Composed of a thin layer of peptidoglycan, No teichoic acid
Gram - negative cell wall
Between outer membrane and gram negative cell wall
Periplasma space
Has a gram positive reaction
Acid-fast cell wall
Concurs resistance to drying and chemicals
Acid-fast cell wall
Contain a waxy layer of phospholipids and fatty acids (hydroxymethoxy acid or mycolic acid) bound to the exterior of the cell wall
Acid-fast cell wall
Acid-fast organisms
“No MILC”
Nocardia
Mycobacterium
Isospora
Legionella micdadei
Cryptosporidium
Other clinically relevant organisms with cell wall containing mycolic acid
Nocardia
Rhodococcus
Gordonia
Tsukamurella
Corynebacterium
Bacteria without cell walls contain ___
Sterols
Examples of bacteria without cell wall
Mycoplasma, ureaplasma
Present only in gram-negative bacteria
outer membrane
Composed of proteins, phospholipids, and lipopolysaccharide
Outer membrane
Lipopolysaccharide is comprised of:
Lipid A
Core polysaccharide
O polysaccharide
Vital to evade the host defenses;
it contributes to the negative charge of the bacterial surface
Lipopolysaccharide
It comprises endotoxin
Lipopolysaccharide
Essential component of both Gram pos and gram neg bacteria
Cell membrane
Has affinity for basic dyes
Metachromatic Granules
Site of bacterial energy metabolism
Cell membrane
Special stains for metachromatic granules
Albert
Neisser
Ponder
Methylene Blue
Babes Ernst Granules
Corynebacterium diphtheriae
“Corny mo, Babe”
Much granules
Mycobacterium tuberculosis
Bipolar granules
Yersinia pestis
“Safety pin appearance”
Function as a survival response to certain adverse nutritional conditions, such as depletion of a certain source
Endospore
Highly resistant to desiccation, heat, and various chemicals
The last structure to be destroyed
Endospores
Basis of QC in autoclaving
Endospores
Endospores are helpful in identifying some species of bacteria like
Bacillus and Clostridium
Terminal spore
Clostridium tetani
Subterminal spore
Clostridium botulinum
Central spore
Bacillus anthracis
Boxcar appearance
Bacillus anthracis
Possess a core that contains many cell components, a spore wall, a cortex, a coat, and an exosporium
Endospore
The core of endospore contains ____, which aids in heat resistance within the core
Calcium dipicolinate
Endospore stains
Schaeffer-Fulton stain (green)
Doemer’s stain
Wirtz-Conklin
True or False.
Endospore germinate under favorable nutritional conditions after an activation process that involves damage to the spore coat. They are not reproductive structures
True
Gives motility to bacteria. For locomotion
Flagella
No flagella
Atrichous
Single flagellum at each pole
Monotrichous
Examples of Monotrichous
Vibrio
Campylobacter
Helicobacter
Pseudomonas aeruginosa
NO Enterobacteriaceae are monotrichous
Single flagellum at each pole
Amphitrichous
Tuft of flagella at one or both poles
Lophotrichous
Flagella all over organism
Peritrichous
Examples of Peritrichous
Enterobacteriaceae, except SKY (Shigella, Klebsiella, Yersinia)
Most common flagella in pathogens
Peritrichous and monotrichous
Demonstrate motility at ____ hours growth
18
Often, motility can be best seen at ____
25 deg C (35-37 deg C may be inhhibitory)
Component of flagellar stains
Tannic acid
Examples of flagellar stains
Leifson
Fisher and Conn
Gray
Precipitates and coats flagella
Tannic acid
AKA axial filament
Found in spirochetes
Endoflagella
Tumbling motility
Listeria monocytogenes
Darting motility
Campylobacter
Gliding motility
Capnocytophaga
Swarming motility
Proteus
Hair-like extensions that extent into the environment
Pili/Fimbrae
Type of pili that serves as an attachment
Common pili
Pili for conjugation
Sex/conjugate pili
“bacterial sex”
Bacteria reproduce by asexual reproduction via
Binary fission
4 phases of the growth cycle
Lag phase
Logarithmnic/exponential phase
Stationary phase
Decline and death
Little or no multiplication but enzymes are very active. A period of adjustment and adaptation
Lag phase
Organisms grow at maximum rate (exponential rate).
Most sensitive to antimicrobials
Logarithmic phase
Plateau-growth ceases because nutrients are exhausted or toxic metabolic products have accumulated
Stationary phase.
Direct microscopic count may remain constant but viable count slowly decreases
Decline and death
3 mechanisms of bacterial gene transfer
- Transformation
- Conjugation
- Transduction
Direct uptake DNA from surrounding environment
-Allows evolution of DNA over time
-Introduce genes to bacteria for replication
Transformation
Transfer from one cell to another via pillus; requires physical contact of 2 organismsm
-DNA transferred via plasmids
Conjugation
Small DNA molecules within a cell
Plasmids
Transfer of DNA via a bacteriophage (virus that infects bacteria)
Transduction
Nuclear material enters bacteria
-Multiplies, lyses cells
-Releases progeny viruses
Lytic cycle
Nuclear material enters cell
-Incorporates in host DNA
-May later become excised then enter lytic phase
-Genes for some bacterial toxins are transferred to non-toxic strains via lysogeny
Lysogenic cycle
Phages that replicate only via the lytic cycle
Virulent
Phages that replicate using lytic cycle and lysogenic cycle
Temperate
“jumping genes”
DNA segments within bacterial DNA
Transposons
A mechanism of transfer of resistance to antibiotics; can even transfer antibiotic resistance genes between 2 different bacterial species
Transposition
Require preformed organic compounds for growth, includes most medical important bacteria
Heterotrophs/Organotrophs
Do not require preformed organic compounds for growth because they can synthesize them from inorganic compounds and carbon dioxide
Autotrophs/Lithotrophs
Use light as energy source
Phototrophs
Use redox potential generated by various chemical reactions
Chemotrophs
Grows in the presence of atmospheric (free) oxygen)
Aerobe
Cannot grow in the absence of free oxygen, which serves as final electron acceptor. Contain superoxide dismutase, which protects from the toxic oxygen
Obligate aerobe
Requires reduced amount of free oxygen (5% O2)
Microaerophile
Grows in the absence of atmospheric oxygen
Anaerobe
Cannot grow in the presence of atmospheric oxygen;
lack superoxide dismutase, catalase, and cytochrome-C oxidase (enzymes that destroy toxic products of oxygen metabolism)
Use nitrates, sulfates, and carbonates as final electron acceptor
Obligate anaerobes
Fundamentally an aerobe but can grow in the absence of atmospheric oxygen
Facultative anaerobe
Does not grow well, but survives in the presence of atmospheric oxygen
Aerotolerant
Obligate Aerobes
Mycobacterium
Corynebacterium
P. aeruginosa
Nocardia
Bacillus
“May Cash sa PNB”
Obligate Anaerobes
Actinomyces
Bacteroides
Clostridium
Fusobacterium
Eubacterium
Prevotella
Aerotolerant
Lactobacillus
Propionibacterium
Microaerophiles
Campylobacter
Helicobacter
Facultative anaerobe
Most pathogenic bacteria are facultative anaerobes
Growth of obligate aerobes in thioglycollate broth
growth at the top
Growth of facultative anaerobes and aerotolerant anaerobe in thioglycollate broth
growth all throughout
growth of obligate anaerobe in thioglycollate broth
growth at the bottom
Growth enhanced by the increased CO2 (5-10% CO2)
Capnophile
Capnophiles
Streptococcus
Campylobacter
Haemophilus
Helicobacter
Moraxella catarrhalis
Mycobacterium
Pathogenic Neisseria
“Sa Camp Here in Heaven, May Money Po!”
Grow below 10 deg C
Ex: L. monocytogenes , blood bank contaminants
Psychrophilic
Grow at 20 - 40 deg C (best at 30 - 37 deg C).
Most pathogens
Mesophilic
grow at 50 -55 deg C (basis of test for effective autoclaving
Thermophilic
Specimen is placed in the refrigerator
-inhibits growth of bacteria except L. monocytogenes and Y. enterocolitica
Cold enrichment technique
Lactobacillus grows in a special medium called __
Tomato juice agar
Vibrio species are alkalophiles and grows in ___
Alkaline Peptone Water: enrichment medium and
TCBS: differential medium
total absence of viable microorganisms as assessed by no growth on any medium
Sterility
kills bacteria
Bactericidal
inhibits growth of bacteria
Bacteriostatic
removal or killing or all microorganisms
sterilization
removal or killing of disease-causing microorganisms (inanimate objects)
Disinfection
any procedure that inhibits the growth and multiplication of microorganisms
Antisepsis
Method of sterilization and disinfection that coagulates protein
Moist heat
100 deg C for 15 - 30 minutes
-kills all vegetative organisms but not all spores or viruses
Boiling
Alternate heating (kills vegetative cells), incubation (spores germinate), heating
Fractionation
Flowing steam for 30 minutes on 3 successive days
Tyndallization
75 - 80 deg C for 2 hours on 3 successive days
Inspissation
Used to sterilize media containing milk or serum
Fractionation
Uses steam under pressure
121 deg C at 15 psi for 15 - 30 minutes
Autoclaving
Most effective method of sterilization
Autoclaving
Used to raise temperature in autoclaving
pressure
Kills organisms including viruses and spores;
Used whenever possible to sterilize liquid media, instruments, glassware
Autoclaving
Quality control in autoclaving
spores of Bacillus stearrothermophilus incubated at 56 deg C
recommended for milk and dairy products
Pasteurization
Low temperature holding
63 deg C for 30 minutes
Batch pasteurization
High temperature short time at 72 deg C for 15 seconds
Flash Pasteurization
Kills by oxidation
Dry heat
Temp and duration for oven using dry heat
160 - 180 deg C for 1 -2 hours
Quality control of dry heat
spores of Bacillus subtilis incubated at 35 - 37 deg C
To sterilize inoculating loop
Flame
Size of membrane filter that has 100% bacterial sterility
0.22 um
For urea broth and sugar fermentation broth
Filtration
UV light
Radiation
Cold sterilization
Used for materials that cannot be autoclaved
Ethylene oxide gas
Quality control for ethylene oxide gas
spores of Bacillus subtilis incubated at 35 - 37 deg C
Destroys vegetative forms on inanimate objects
Disinfectant
denature proteins; activity reduced by organic matter (cresol less affected)
Phenolics
Inactivate bacteria by binding with their hydrophobic and lipophilic groups, interacting with the cell membrane to alter metabolic properties and permeability
Quaternary ammoniums (quats)
oxidizes organic matter
Chlorine
safe for delicate lensed instruments; usually poor activity against Pseudomonas
2% aqueous glutaraldehyde
a dipheyl cationic analog that is useful for topical disinfectant
Chlorhexidine
5 types of disinfectants
- Quaternary ammoniums
- Phenolics
- Chlorine
- 2% aqueous glutaraldehyde
- Chlorhexidine
Phenol is used as a disinfectant standard that is expressed as a ___.
This compares the rate of the minimal sterilizing concentration of phenol to that of the test compound for a particular organism
phenol coefficient
The endpoint of phenol coefficient is the lowest concentration that kills test organisms in ___
10 minutes at 20 deg C
Test organisms for phenol coefficient
Salmonella typhi and Staphylococcus aures
agent used on living tissue
antiseptic
3 types of antiseptics
- Alcohol
- Tincture (alcoholic) iodine
- Iodophor
__ is necessary for protein denaturation
Water
___ is the major form of alcohol used in hospitals
Isopropyl alcohol (90-95%)
Tincture (alcoholic) iodine is composed of:
2% solution of aqueous alcohol containing potassium iodide
Iodophor is composed of:
Iodine + detergent (e.g. povidone-iodine)
Biologic indicators
1. Autoclave:
2. Ionizing radiation:
3. Dry heat oven:
4. Ethylene oxide:
Biologic indicators
1. Autoclave: Bacillus stearothermophilus
2. Ionizing radiation: Bacillus pumilis
3. Dry heat oven: Bacillus subtilis var niger
4. Ethylene oxide: Bacillus subtilis var globigii
Designed to protect the operator, the laboratory environment, and work materials from exposure to infectious aerosols and splashes that may be generated when manipulating materials containing infectious agents
Biological safety cabinets
Open front BSC. Has HEPA filter. Filtration happens once.
Provides minimal personnel protection. Does not protect work surface
Class I BSC
Laminar flow cabinets with variable sash opening. Filtration happens twice. Most commonly used in hospital micro labs. Provides protection for worker and work surface
Class II BSC
Completely enclosed. Negative pressure. has gloves. Provides maximum protection
Class III BSC
Biosafety level not known to cause disease in healthy adults
Level I
Biosafety level: common pathogens
Level 2
Biosafety level: causes serious lethal disease via inhalation. Effective treatment available
Level III
Biosafety level that causes life-threatening disease. No vaccine
Level IV
Found in the cell wall of S. aureus
-inhibits phagocytosis by binding Fc portion of IgG
Protein A
Enzymes that cleave IgA,
allows colonization of mucosal surfaces
-produced by S. pneumoniae, H. influenzae, N. gonorrhoeae, N. meningitidis
“SHiNe”
IgA protease
Found as a surface molecule of group A streptococcus,
prevents phagocytosis
M protein
Only in gram neg bacteria
-lipopolysaccharide
-released when bacteria die
-heat resistant
Endotoxin
Like LPS but lacks O antigen
Endotoxin activity
Found on non-enteric gram negs (N. meningitidis is most important example)
Lipooligosaccharide
Sore throat with membrane, swollen nodes
-Inactivates elongation factor (EF-2), which is necessary for protein synthesis.
Corynebacterium diphtheriae
Causes many types of infection (skin infections, sepsis, pneumoniae)
-same mechanism as diphtheria toxin
Pseudomonas aeruginosa
(Exotoxin)
Causes infectious diarrhea
-invasion of GI mucosal cells is still the main cause of diseases;
-nontoxigenic strain causes significant disease
Shigella
(Shiga toxin)
Strain of E. coli that produce “shiga-like” toxin;
Typically cause bloody diarrhea
-can also cause hemolytic uremic syndrome (HUS) when toxin reaches system circulation
-Classic serotype is E.coli O157:H7
Enterohemorrhagic E. coli (EHEC)
Shiga-like toxin
aka Traveller’s diarrhea
-causes watery diarrhea
Enterotoxigenic E. coli (ETEC)
Heat labile toxin (LT)
Heat stable toxin (ST)
3 proteins of Anthrax
Protective antigen (PA)
Lethal factor (LF)
Edema factor (EF)
2 proteins of B. anthracis that causes edema toxin
Protein antigen
Edema factor
Causes “rice-water” diarrhea
that leads to dehydration, electrolyte losses, and shock
Vibrio cholerae
(Cholera toxin)
Causes whooping cough
Bordetella pertusis
Preferred specimen for the determination of Bordetella pertusis (whooping cough)
Nasopharyngeal swab
Works in spinal cord (Renshaw cells)
- results in spastic muscle paralysis
Clostridium tetani
(Tetanospasmin)
Works at neuromuscular junctions
-results in muscles floppy (Flaccid paralysis)
-In babies: Floppy Baby Syndrome ( contaminated honey)
Clostridium botulinum
(Botulinum toxin)
Causes Gas Gangrene
Phospholipase C enzyme
Clostridium perfringens
(Alpha toxin)
“Cytolysin” (lysis cells)
-Responsible for beta hemolysis
-Trigger production of anti-streptolysin O (ASO)
-Can be usefu lin suspected rheumatic heart disease or post strep glomerulonephritis
Streptococcus pyogenes (Streptolysin O)
Activate a MASSIVE number of T-cells (2-20% T cells)
Superantigens
2 examples of superantigens
TSST-1 (S. aureus)
Pyrogenic exotoxin A or C (S. pyogenes)
Reagents in Gram staining
- Primary stain - Crystal Violet
- Mordant - Gram’s Iodine
- Decolorizer - Alcohol-Acetone
- Secondary stain - Safranin
“VIAS”
Bacteria with thick cell walls containing teichoic acid retain the crystal violet-iodine complex dye after decolorization.
Gram Stain
Primary stain binds to mycolic acid in the cell walls of mycobacteria and is retained after the decolorizing step with acid-alcohol
Acid-Fast Stain
Reagents in AF Staining
- Primary Stain - Carbol fuchsin
- Mordant - Heat or phenol
- Decolorizer - Acid-alcohol
- Methylene Blue
Size of AFB smear
at least like a thumb size or
2 cm x 3cm
Fluorochrome dye that stains both gram positive and gram negative bacteria, living or dead.
-Binds to the nucleic acid of the cell and fluoresces as a bright orange when fluorescent microscope is used.
Acridine Orange
Fluorochrome that binds to chitin in fungal cell walls.
-Fluoresces as a bright apple-green or blue-white, allowing visualization of fungal structures with a fluorescent microscope
Calcoflour White
Traditionally has been used to stain C. diphtheriae for observation of metachromatic granules
Methylene Blue
Used to stain the cell walls of medically important fungi grown in slide culture
Lactophenol Cotton Blue
Negative stain used to visualize capsules surrounding certain yeasts, such as Cryptococcus spp
India Ink
Primary stain: malachite green (heated to steaming for about 5 minutes)
Counterstain: Safranin
Endospores appear green with pink-appearing or red-appearing bacterial cells
Endospore stain
Fluorescein-labeled antibodies made from antisera produced by injecting animals with whole organisms or complex antigen mixtures
- Resultant polyclonal antibodies may react with multiple antigens on the organism that was injected and may also cross-react with antigens or other microorganisms or possibly with human cells in the specimen
Immunofluorescent antibody (IF) staining
IF staining is useful in confirming the presence of specific organisms such as
Bordetella pertusis or Legionella pneumophila
Percentage of agar in semi-solid media
0.5 - 1%
Percentage of agar in liquefiable solid media
2-3%
Tissue culture system from African green monkey
Vero cell
Tissue culture system from mouse cell line
Mccoy cell
Tissue culture system from Lung carcinoma
A549 cell
Tissue culture system from Cervical carcinoma
Hela cell
Tissue culture system from laryngeal carcinoma
Hep-2 cell
Supports the growth of non-fastidious organism
-Nutrient agar or broth; Trypticase soy agar or broth
Simple or General Purpose
Select the growth of particular organism and prevents other organism thru inhibitors
Selective
Selective medium for Mycobacterium tuberculosis
Lowenstein Jensen
Selective medium for Gram positive microorganism
Phenylethyl alcohol
Selective media for Neisseria gonorrhea
Thayer Martin, Martin Lewis, New York City
Culture media that contains nutrient supplements
Enriched
Enriched media that contains 5% defibrinated Sheep blood (Horse/rabbit)
Blood agar plate
Enriched medium for Haemophilus
Chocolate agar plate
Preparation of Chocolate agar plate
-Add blood while base medium is still hot to lyse the RBCs
-Release X and V factors
X factor
Hemin
V Factor
NAD
Can enhance the growth of microorganism
Enrichment
Enrichment medium for Vibrio
Alkaline Peptone Water
Enrichment media for Salmonella and Shigella
Selenite and Tetrathionate
Allow certain types of organisms to grow and differentiate, and inhibit the growth of other organisms
Selective-Differential
Selective-Differential medium for S. aureus and S. epidermidis/saprophyticus
Mannitol Salt Agar
Selective-Differential medium for Vibrio spp
Thiosulfate Citrate Bile Salt
Provides distinct colonial appearance of microorganisms to know their identification
Differential
Differential media that contains 3 sugars (lac, gluc, suc)
Triple Sugar Iron
Standard medium for specimens
Blood agar (usually made with 5% sheep blood)
Hemolysis with green zone around colony. May be narrow or wide. Partial lysis of RBCs
Alpha hemolysis
Complete lysis of RBCs. Clear zone around colony.
Beta hemolysis
No lysis of RBCs. No zone of hemolysis
Gamma
Examples of organisms that are alpha hemolytic
Streptococcus pneumoniae
Viridians streptococci
Some enterococci
Examples of organisms that are beta hemolytic
Group A strep
Group B strep
Listeria monocytogenes
Examples of organisms that are gamma hemolytic
Some enterococci
___ is readily identified in the laboratory by its characteristic “double zone” hemolysis also known as target hemolysis
Clostridium perfringens
Small area of intact RBCs around colony surrounded by a wider zone of complete hemolysis
Alpha-prime or wide zone hemolysis
Enriched medium that will grow most nonfastidious bacteria
Sheep blood agar
What is added to the blood agar medium to allow differentiation of hemolysis
Tryptic soy agar with 5% sheep blood
Enriched medium for Haemophilus and Neisseria. Supplies X and V factors
Chocolate agar
Selective medium for gram-positive microorganisms
Columbia colistin nalidixic agar (CNA)
*CN suppress most GN
Selective medium for gram-positive cocci and anaerobic gram-negative rods
Phenylethyl alcohol agar (PEA)
*inhibits enteric gram-negative rods.
Selective medium for S. pyogenes and S. agalactiae
Streptococcal selective agar (SSA)
*contains trimethoprim-sulfamethoxazole
Selective differential medium for isolation of enteric gram-negative rods
Eosin methylene blue (EMB)
*EM inhibit gram positives
*Lactose fermenters are green-black or purple
*Nonlactose fermenters are colorless
In EMB, E. coli produces ___
Green metallic sheen
Selective, differential medium for isolation of enteric, gram-negative rods
MacConkey Agar (MAC)
*Bile salts and crystal violet inhibit most gram positives
*Lactose fermenters are pink
*Non-lactose fermenters are colorless
Selective, differential medium for E. coli O157:H7
MacConkey sorbitol agar (SMAC)
*E. coli O157:H7 does not ferment sorbitol. colonies are colorless
Selective, differential medium for isolation of enteric pathogens from stool
Hektoen enteric agar (HE)
*Bile salts, bromthymolblue, acid fuschin inhibit normal GI flora
*Non pathogens are orange to salmon pink
*Nonlactose fermenters are green to blue green
*H2S positive colonies have black precipitate
Selective, differential medium for isolation of Salmonella and Shigella in stool
Xylose lysine deoxycholate (XLD)
*Deoxycholate inhibits gram neg rods and gram pos
Four types of colonies in XLD
-Yellow (E. coli)
-Yellow with black centers (Some Proteus spp)
-Colorless or red colonies (Shigella)
-Red colonies with black centers (Salmonella)
Selective medium for Salmonella and Shigella
Salmonella-Shigella agar (SS)
*Brilliant green and bile salts inhibit coliforms
*Salmonella and Shigella do not ferment lactose - colonies are colorless
*Salmonella produces H2S
Selective enrichment medium for isolation of Salmonella and Shigella
Gram-negative broth (GN)
*Deoxycholate and citrate salts retard growth of gram-positives
Enrichment broth used for recover of Salmonella from stool
Selenite broth
*Subculture onto selective differential agar after 6-8 hours of incubation
Enrichment broth for recovery of Salmonella from stool
Tetrathionate broth
*Bile salts and sodium thiosulfate inhibit gram positives and Enterobactericeae.
Selective enrichment medium for isolation of Campylobacter from stool
Campylobacter blood agar (Campy BAP)
*Incubate pates in increased CO2 at 42 deg C
selective enrichment medium for recovery of Neisseria gonorrhoeae and Neisseria meningitidis from specimens with normal flora
Modified Thayer Martin (TM)
Antibiotics in Modified Thayer Martin (TM)
VCNT
Vancomycin
Colistin
Nystatin
Trimethoprim (inhibits swarming of Proteus)
Thayer Martin antibiotics
VCN
Selective enrichment medium for recovery of N. gonorrhoeae and N. meningitidis from specimens with normal flora
Martin Lewis
Antibiotics is Martin-Lewis
VCAT
*Anisomycin - antifungal
Selective enrichment medium for isolation of N. gonorrhoeae and N. meniningitidis from specimens with normal flora
New York City medium (NYC)
Antibiotics used in NYC medium
VCAbT
*Amphotericin B: Antifungal
Antibiotics to inhibit GN and GP bacteria and yeast
GC-LECT
For transportation and growth of N. gonorrhoeae
JEMBEC plates
Selective differential medium for isolation and identification of Bacteroidesfragilis
Bacteriodes bile-esculin agar (BBE)
Enrichment medium for isolation of fastidious anaerobes
Blood agar, anaerobic, CDC
Selective medium for Clostridium difficile
Cycloserine cefoxitin fructose egg yolk agar (CCFA)
For isolation of anaerobes, especially pathogenic Clostridium
Cooked meat medium
For determination of lecithinase and lipase production by clostridia and fusobacteria
Egg-yolk agar (EYA)
Selective medium for Bacteroides and Prevotella
Laked kanamycin-vancomycin blood agar (LKV)
Inhibits enteric gram-negative rods and swarming by some clostridia
Phenylethyl alcohol agar (PEA)
All-purpose medium that supports the growth of most aerobes and anaerobes. Can be used as a back up broth to detect organisms present in small numbers or anaerobes
Thioglycollate broth (THIO)
In thioglycollate broth, what acts as a reducing agent?
Thioglycolate
*Aerobes grow at top, strict anaerobes at bottom, facultative anaerobes throughout.
Storage of thioglycollate broth
Store at room temperature and boil and cool prior to use
Differential medium for isolation of Corynebacterium diphtheriae
Cystine-tellurite blood agar
Enrichment medium for recovery and identification of C. diphtheriae
Also promotes development of characteristic granules
Loeffler medium
Selective differential medium for C. diphtheriae
Tindale agar
Selective medium for isolation of Salmonella
Bismuth sulfite agar
Selective medium for Yersinia enterocolitica, Aeromonas, and Plesiomonas shigelloides
Cefsulodin-Irgasan-Novobiocin agar (CIN)
___ ferments manitol and appears red “bull’s eye” colonies surrounded by colorless halo in CIN agar
Y. enterocolitica
Enrichment medium for recovery of Vibrio and Aeromonas from stool
Alkaline Peptone Water
Selective medium for Vibrios
TCBS
Used to speciate Haemophilus
Rabbit Blood Agar
Selective enrichment medium for isolation of Bordetella pertusis
*Cough plate
*Colonies resemble mercury droplets
Bordet-Gengou
Selective for B. pertusis
Regan Lowe
Enrichment medium for isolation of Legionella
Buffered charcoal yeast extract agar (BCYE)
Nonselective enrichment medium for isolation of Garnerella vaginalis
Vaginalis agar (V agar)
Semi-selective medium for G. vaginalis
Human blood Tween agar (HBT)
Selective enrichment medium used to culture mycobacteria
*Colonies are rough and buff
Lowenstein-Jensen
Selective enrichment medium used to culture mycobacteria
* Isoniazid-resistant strains grow better
Middlebrook 7H10 and 7H11
Enrichment medium for isolation of Leptospira from blood, CSF, and urine
Fletcher medium
Catalase-Positive organisms
Nocardia
Pseudomonas
Listeria
Aspergillus
Candida
E. coli
Staphylococci
Serrati
B. cepacia
H. pylori
“Cats Need PLACESS to Belch their Hairballs”
Slide test is a screening test. Detects bound coagulase (clumping factor)
If negative, tube test should be performed. Detects free coagulase
Coagulase test
Agglutination of latex beads coated with fibrinogen and antibodies to protein A (protein in cell wall of S. aureus)
Slide agglutination tests for S. aureus
test used for S. saprophyticus vs other CONS
Novobiocin
“NO SRES”
Detect DNase activity in aerobic bacteria
DNase test
Selective and differential for Staphylococcus species
Mannitol Salt agar (7.5% salt)
Identify group A and group B beta hemolytic streptococci
Bacitracin- SXT Susceptibility
Bacitracin and SXT results of
Group A strep:
Group B strep:
Not Group A /Group B:
Group A strep: S - R
Group B strep: R - R
Not Group A /Group B: R - S
“B-BRAS”
Differentiate Enterococcus (+) spp from non Enterococcus spp (-)
Group A step are also +
(+): pink to cherry-red color after the addition of the color developer
PYRase test
Identify group B streptococcus
Group B strep produces extracellular protein.
Characteristic “arrowhead” hemolytic pattern results when S. agalactiae is streaked perpendicularly to beta hemolytic S. aureus
CAMP
Identify Group B streptococcus
Alternative to CAMP
(+): purple color after adding ninhydrin reagent
Hippurae hydrolysis
Lancefield grouping of streptococci and enterococci (based on the antigenic nature of cell wall carbohydrates)
Slide agglutination tests
Differentiate S. pneumonia (S) from other alpha hemolytic streptococci (R)
Optochin
“OVRPS”
Optochin is also known as P disk. It contains:
Ethyl hydrocuprein hydrochloride
Identification of S. pneumoniae (+)
Bile salts cause lysis of some organisms
(+): clearing of broth or disappearance of colony
Bile solubility
Differentiate enterococcus (+) species from non-Enterococcus species (-)
Group A strep are also (+)
(+): Group D Enterococcus
“DE sa Pyr”
PYRase Test (PYR Test)
Distinguish Group D Streptococcus and Enterococcus from other Lancefield groups
Group D strep can grow in 40% bile and hydrolyze esculin to form esculetin.
Esculetin reacts with ferric citrate to form a brown-black precipitate
Bile-Esculin Agar
Differentiate group D streptococci from enterococci
6.5% Salt Broth (Salt Tolerance)
Differentiate carbohydrates in CTA tubes are oxidized into organic acids that change phenol red into a yellow color
*Differentiate Neisseria species
Cystine Trypticase Agar (CTA) Sugars
Sugar in N. gonorrhoeae
N. meningitidis
N. lactamica
N. sicca
N. elongata
M. catarrhalis
Glucose
Gluc, Maltose
Gluc, Maltose, Lac
Gluc, Maltose, Sucrose
(no sugar)
(no sugar)
Good test to differentiate Enterobacteriacea from nonfermeters.
Cytochrome oxidase, in the presence of atmospheric oxygen to form a colored compound, indophenol
(+): Maroon-violet color within 30 seconds
Cytochrome oxidase
Aids in identifying members of Enterobacteriaceae (mostly +)
-If organism reduced nitrates, red color develops
Nitrate reduction
Differentiate Enterobacteriaceae members
All Enterobacteriaceae ferments glucose
-With phenol red indicator, change from red to yellow is positive result
Carbohydrate Fermentation
Test for slow lactose fermentation
Helpful in differentiating Citrobacter (+) from most Salmonella (-)
-Changed to orthonitrophenol by beta-galactosidase
ONPG
Good test to differentiate Salmonella (+) from Shigella (-)
-H2S reacts with iron salt in medium to form black ferrous sulfide
H2S production
Differentiate Enterobacteriaceae members
-Sugar fermentation produces acid, changes color of pH indicator
Triple Sugar Iron (TSI) agar
Examples of microorganisms with K/A TSIA result
Salmonela, Shigella, P. mirabilis, Citrobacter
Examples of microorganisms with A/A TSIA result
E. coli
Klebsiella
Enterobacte
“EKE”
P. vulgaris, Citrobacter
Examples of microorganisms with K/K TSIA result
P. aeruginosa
Never enterobacteriaceae
Distinguish enterobacteriaceae.
Tryphtophan inthe medium is oxidized by certain bacteria which produce tryptophanase.
Indole reacts with p-dimethylaminobenzaldehyde to form a red product
Indole test
Indole positive organisms
E. coli
P. vulgaris
K. oxytoca
Differentiate lactose fermenting enterobacteriaceae using 40% KOH
Methyl Red- Voges Proskauer
MR positive microoragnism
E. coli
VP positive organisms
Klebsiella
Enterobacter
Serratia
Hafnia
“Si VP namigay ng KESH”
Identification of lactose-dermenting enterobacteriaceae
Converts into ammonia, which is then converted to ammonium hydroxide.
the rise in pH causes a change in the color of the bromthymol blue indicator
Citrate
Distinguish enterobacteriaceae. Causes color change of phenol red from light orange at pH 6.8 to magenta pink at pH 8.1
Urease
Urease positive microorganisms
Proteus
Cryptococcus
H. pylori
Ureaplasma
Nocardia
Klebsiella
S. epidermidis
S. saprophyticus
“Pee CHUNKSS”
Distinguish enterobacteriaceae wherein phenylaline deaminase deaminates phenylalanine to phenyl pyruvic acid, which reacts with ferric chloride to produce green color
Phenylalanine deaminase
Microorganisms positive for PD
Proteus
Providencia
Morganella
PD-PPMo-Green
IF an organisms has enzyme to decarboxylate amino acid
Distinguish enterobacteriaceae
Decarboxylase reactions
Medium used contains a small amount of agar.
Motility
2 organisms that are the only nonmotile Enterobacteriaceae
Shigella and Klebsiella
This test is used to determine the ability of an organism to use acetamide as the sole source of carbon
Acetamide Utilization Test
Organism positive for Acetamide Utilization test
Pseudomonas aeruginosa
This test is used to determine if an organism can use acetate as the sole source of carbon
Acetate Utilization Test
Positive microorganism for acetate utilization test
E. coli
The only indole positive Enterobacteriaceae
K. oxytoca
Lactose negative Enterobacteriaceae
Shigella
Edwardsiella
Salmonella
Citrobacter
Serratia
Proteus
Morganella
Providencia
Yersinia
H2S positive Enterobacteriaceae
Proteus
Edwardsiella
Citrobacter
Salmonella
“PECS”
VP Positive Enterobacteriaceae
Klebsiella
Enterobacter
Serratia
Hafnia
“Hafnia”
PD Positive Enterobacteriaceae
Proteus
Providencia
Morganella
Klebsiella (slow)
Urease positive Enterobacteriaceae
Pee CHUNKSS
Nonmotile at 35 deg C Enterobacteriaceae
Shigella
Klebsiella
Yersinia (motile at 22 deg C)
Enterobacteriaceae Lactose Fermenters
Escherichia
Klebsiella
Enterobacter
EKE
Enterobacteriaceae that are Late Lactose Fermenters
Citrobacter
Salmonella arizona
Shigella sonnei
Serratia
Hafnia
Yersinia
Enterobacteriaceae that are non-lactose fermenters
Proteus
Providencia
Morganella
Erwinia
Salmonella
Shigella
Edwardsiella
“PPMo SESE”
There is an inverse linear relationship between the diameter of the zone of inhibited growth around the antibiotic disk and the logarithm of the minimal inhibitory concentration (MIC) of the antibiotic
Kirby-Bauer Disk Diffusion Test
Kirby-Bauer Disk Diffusion uses ___ agar
Mueller-Hinton agar
size of the MHA plate
150 x 15 mm plate
pH of the MHA
7.2 - 7.4
MHA depth
4 mm (some sources 4-6 mm)
Density of the inoculum should be adjusted to ___ standard
0.5 McFarland standard (equivalent to 1.5 x 108 CFU/mL
Composition of 0.5 McFarland standard
0.5 ml of 1.175% BaCl2 and 99.5 ml of 1% H2SO4
Only overnight colonies less than ___ hours in Kirby-Bauer Disk Diffusion test should be tested
24 hours
IN the AST, let the inoculum dry at least ___ minutes but not more than ___ minutes
5 minutes; 30 minutes
Maximum number of disks per 150 mm plate
12 disks
(8 disks in outer ring, 4 disks in center)
Apply antibiotic disks, tap down firmly, and wait ___ at room temperature to allow diffusion in to the medium
3-5 minutes (not more than 15 minutes)
2 antibiotics that are best indicators of poor storage , as they are the first to deteriorate
Penicillin and methicillin
Incubate AST plate at __
35 deg C for 16 to 18 hours
Used to measure zone of inhibition with the unaided eye to the nearest mm using ___
Ruler, caliper or template
Test QC strains of E. coli, S. aureus, P. aeruginosa, E. faecalis for 20 - 30 consecutive days.
If results are acceptable, frequency can reduced to ___
Weekly
3 types of beta lactamase direct test
Acidometric
Iodometric
Chromogenic
Chromogenic test that consists of a paper disk impregnated with a chromogenic cephalosporin. If the organism is producing beta lactamase, hydrolysis of the chromogenic cephalosporin will occur and the disk color will change from yellow to red, usually within a minute
Beta lactamase testing
Standard inoculum added to serial dilutions of antibiotics and incubated.
The least amount of antibiotic (highest dilution) that prevents visible growth
Minimal inhibitory concentration (MIC)
Concentration of antibiotic that results in 99.9% decreased in CFU/mL
Minimal bactericidal concentration (MBC)
Serial dilutions of patient’s peak and trough specimens inoculated with standardized amount of patient’s pathogen and incubated overnight
Serum bactericidal test (Schlicter test)
Test with oxacillin disk instead of penicllin. more sensitive in detecting resistance
Screen fro penicillin susceptibility in S. pneumoniae
Oxacillin used as class representative for pencillinase-resistance penicillins. Organisms resistant to it is resistant to all.
Tests fro oxacillin- (methicillin)- resistant - S. aureus
For detection of vancomycin-resistant enterococci
Vancomycin screen
Plastic strip containing antibiotic concentration gradient placed on inoculum lawn on MH plate and incubated overnight.
Useful for testing fastidious organisms such as S. pneumoniae, other strep, H. influenae and anaerobes
E test
TO detect inducible clindamycin resistance in MRSA isolates that are resistant to erythromycin and susceptible to clindamycin on initial testing.
D test
Aerobic Gram positive cocci
Micrococcus
Enterococcus
Streptococcus
Staphylococcus
“MESS”
Anaerobic gram positive cocci
Peptococcus
Peptostreptococcus
Sarcina
“PPS”
Aerobic Gram positive bacilli
Bacillus
Corynebacterium
Erysipelothrix
Lactobacillus
Listeria
Mycobacterium
Nocardia
“BCELLMN”
Anaerobic Gram positive bacilli
Actinomyces
Clostridium
Cutibacterium (Propionibacterium_
“ACC”
Aerobic gram negative cocci
Moraxella catarrhalis
Neisseria
Anaerobic gram negative cocci
Veillonella
Anaerobic gram negative bacilli
Bacteriodes
Fusobacterium
Pleomorphic
Mycoplasma, Ureaplasma
Anaplasma
Chlamydia
Erhlicia
Rickettsiae
Spirochetes
Borrelia
Leptospira
Treponema
“BoLET”
Separates Staphylococcus (and Micrococcus) from Streptococcus
Catalase test
Why are colonies from blood agar not used in catalase test?
Blood has catalase and will give a false positive result
-Use tryptic soy agar instead
-Gram positive spherical cells, usually arranged in grapelike clusters
-Non-motile and non-spore forming
-Grow on many types of media and are active metabolically
-Catalase positive
-Fermentative (produce acid in glucose anaerobically)
-Microdase negative
-Resistant to bacitracin
-Suspectible to furazolidone and lysostaphin
Staphylococcus
Opportunistic pathogen. Infects prosthetic devices
S. epidermidis
2nd most common cause of UTI in young sexually active females
“Honeymooner’s cystitis”
Nitrite negative on urine dipsticks
S. saprophyticus
Often contaminant in blood culture
S. epidermidis
Caused by ingestion of enterotoxin, a preformed toxin.
Classically associated with mayonnaise in potato or egg salad
Staphylococcal Food Poisoning
microorganism that causes toxic shock syndrome using TSST-1. Most common cases are in menstruating women, especially those who are using tampons
S. aureus
Newborn disease caused by S. aureus exfoliative tocxin (Exfoliatin)
Scalded skin syndrome
Superficial cutaneous infection characterized by crusty (honey-crusted) lesions and vesicles surrounded by a red boarder.
Bullous type - caused by S. aureus or S. pyogenes
Impetigo
Causes lobar pneumonia;
Classically occurs following influenza (“post-infectious”)
Pneumonia
Rapid onset of symptoms in patients with NO pre-existing valve disease (unlike subacute bacterial endocarditis)
Acute bacterial endocarditis
Common cause of osteomyelitis
S. aureus
Formerly called enterotoxin F
Toxic shock syndrome toxin - 1 (TSST-1)
Converts hydrogen peroxide into water and oxygen
Catalase
Spreading factor
Hyaluronidase
Fibrinolysin
Staphylokinase
-May be seen in pairs or chains
-Non pigmented colony
-Facultative anaerobes
-Hemolytic or non-hemolytic
Streptococcus
Oxygen stable, non antigenic
Streptolysin O
Oxygen labile, antigenic
Streptolysin O
causes pharyngitis, cellulitis, impetigo (“Honey-crusted” lesions), erysipelas
Pyogenic
15 - 30% pharyngitis are due to ___
S. pyogenes
Scarlet fever, toxic shock-like syndrome, necrotixing fascitis
Toxigenic Strep
Rash following pharyngitis
-skin reaction to erythrogenic toxin
-“sandpaper” skin
-strawberry tongue
Scarlet fever
“Flesh eating disease”
-Infection of deep tissues
-streptococcal pyrogenic exotoxin
Necrotizing fasciitis
Follows group A strep pharyngitis infection
-anti strep antibodies cross react with tissue antigens
-Type II autoimmune reaction
Rheumatic fever
Nephritic syndrome 2-3 weeks after GAS infection
Glomerulonephritis
Indicate recent S. pyogenes infection
ASO titer or anti-DNAse B antibodies
Test susceptibility for scarlet fever
(+) redness or erythema of test site
Dick’s test
Diagnostic test for current scarlet fever
(+) blanching phenomenon (rash fades at site of injection of anti-erythrogenic toxxin)
Schultz- Charlton Reaction
-Colonizes vagina
-causes pneumonia, meningitidis, sepsis, mainly in babies
-most are resistant to bacitracin
-Bile esculin (-)
-PYR (-)
-confirm by precipitin (Lancefield) FA tests or Coagglutination tests
-CAMP (+)
-Hippurate hydrolysis (+)
Group B Beta hemolytic streptococci
Major pathogen of the newborn
-screen pregnant women 35 - 37 weeks of gestation with rectal and vaginal swabs
-patients with positive cultures receive intrapartum penicillin prophylaxis
Group B Beta hemolytic streptococci
Lancet-shaped encapsulated diplococci
Considered part of normal flora of the URT of preschool children
Produces IgA protease
Streptococcus pneumoniae
Principal virulence factor of S. pneumoniae
Antiphagocytic capsular polysaccharide
Lobar pneumonia - associated with “rusty” sputum
S. pneumoniae
Most common caus of bacterial pneumonia in elderly as well as in patients with underlying disease
S. pneumoniae
Most common cause of bacterial meningitis in adults
S. pneumoniae
On culture, appear as alpha-hemolytic, mucoid colonies with flattened or depressed centers
S. pneumoniae
Oropharyngeal commensal; opportunistic pathogens of low virulence.
-subacute bacterial endocarditis at damaged heart valves
-dental caries
Viridians streptococci
Normal colonic flora
E. faecalis and E. faecium
Causes UTI (associated with catheters), biliary tract infections and subacute endocarditis (following GI/GU procedures)
Enterococcus
Important cause of nosocomial infection
Vancomycin-resistant enterococci (VRE)
Relatively resistant to cell wall agents (penicillin, ampicillin, vancomycin)
-PYR (+)
-Variable hemolysis
Enterococcus
Normal colonic bacteria
-Rarely can cause bacteremia and subacute endocarditis
-Strongly associated with colon cancer
Streptococcus bovis (Streptococcus gallolyticus)
-Oval gram-positive cocci in chains
-Usually beta hemolytic on SBA
-Bacitracin (S)
-SXT (R)
- PYR (+)
Group A streptococci (GAS)
-Oval gram positive cocci in chains
-Narrow zone of diffuse beta hemolysis
- SXT and Bacitracin (R)
-Sodium hippurate (+)
-CAMP (+)
Group B Streptococci (GBS)
-Oval, gram positive cocci in chains
-Usually non hemolytic
-Hydrolyzes esculin
Group D streptococci, nonenterococci
-Oval, PC in pairs and chains
-Usually alpha or nonhemolytic
-Hydrolyzes esculin
-Grows in 6.5% NaCl broth
-PYR positive
Enterococcus
-Football-shaped (lancet-shaped) GPC. Usually in pairs. May be single or in short chains. Frequently encapsulated.
-Bile solubility (+)
-Optochin (S)
Streptococcus pneumoniae
-GPC
-Usually alpha hemolytic
-Optochin (R)
-Bile solubility (-)
Viridans streptococci
-GC in pairs, chains
-Butterscotch or caramel odor on SBA
-Usually Lancefield group F
-PYR (-)
-VP (+)
-require 10% CO2
-Grow better anaerobically
Microaerophilic
Aerobic, gram-positive spore-forming rods;
-catalase (+)
Most (but not all) are motile
Bacillus
The only true pathogenic Bacillus
-domesticated animals are the main host
-Causes anthrax
B. anthracis
Most common type of anthrax
-characteristic black painless eschar (ulcer) at the site of inoculation with surrounding edema;
can lead to sepsis
Cutaneous anthrax
Antiphagocytic virulence factor of B. anthracis
Poly-d-glutamic acid capsule
EF + PA
Responsible for cell and tissue edema
Edema toxin
LF + PA
Cause of death in infected animals and humans
Lethal toxin
-Non motile, nonhemolytic on SBA at 24 hours
-Encapsulated (from px)
-Spore-forming cells stain with bamboo pole arrangement
-Rounds up with media in penicillin (“Strings of pearls test”)
-“Medusa Head” colonies on blood agar
-“Boxcar” appearance (central spores)
B. anthracis
could cause food poisoning
B. cereus
Associated with fried rice, milk and pasta
Caused by ingestion of preformed emetic toxin
Emetic type of B. cereus
Associated with meat dishes and sauces, ingested spores develop into vegetative cells that secrete enterotoxins
Diarrheal type of B. cereus
Diphteria bacillus/ Kleb Loeffler’s bacillus
Corynebacterium diphtheriae
An acute, contagious disease characterized by the production of a systemic toxin and a false membrane lining on the throat (pseudomembranous)
Diphteria
-Non-motile
-Nitrate reduction (+)
-Urease (-)
-Catalase (+)
-Glucose and maltose fermented
-Sucrose not fermented
C. diphtheriae
Carried by beta-prophage
-“Lysogenic” phage –> incorporates DNA into bacteria
Diphtheria exotoxin
C. diphtheriae has a direct smear with ___
Loffler’s methylene blue
(culture on Loeffler serum or Pai coagulated egg medium)
Arranged in palisades (“picket fence”) or “Chinese letters” (snapping)
-Metachromatic granules (Babes-Ernest granules)
Corynebacterium diphtheriae
Special culture media required for C. diphtheriae
Loeffler’s or Tinsdale (Tellurite plate)
Media to enhance pleomorphisms and granule production of C. diphtheriae
- Loeffler serum agar (+) poached egg colonies
- Pai coagulated egg medium
Skin test for C. diphtheria susceptibility
Schick’s test
In vitro virulence test for toxin production of C. diphtheriae
-Soak a paper strip in diphtheria antitoxin (100U/mL)
-Produce thin lines of precipitate
Elek method
The paper strip in Elek method is soaked in ____
Diphtheria antitoxin (100U/ml)
In Elek method (C. diphteriae), what is produced by the toxin and antitoxin at 45 degrees angle
Thin lines of precipitate
Nonlipophilic coryneform bacteria
C. pseudodiphtheriticum (Hoffman’s bacillus)
Normal flora of the human nasopharynx
C. pseudodiphtheriticum
C. pseudodiphtheriticum characteristics
Nitrate (+), and urea hydrolysis (+)
Can cause disease similar to clinical diphtheria;
carriage in domestic pets
C. ulcerans
Corynebacterium spp that rarely cause diseases in humans
C. pseudotuberculosis
Other Corynebacterium spp isolated from clinical material
C. xerosis
C. striatum
C. minutissimum
C. amycolatum
Growth is enhanced by lipids
Lipophilic coryneform bacteria
Common cause of diphtheroid prosthestic heart valve endocarditis;
multiple antibiotic resistance allows survival in hospital setting
C. jeikeium
Slowly growing species that multiply resistant to antibiotics
Urease (+)
UTI in immunocompromised patients
Produces alkaline urine and crystal formation
C. urealyticum
Gram (+) or gram variable coccobacillus (resembling Haemophilus), or diphteroid-like rods
Listeria monocytogenes
Facultative intracellular
Catalase positive
Tumbling motility on hanging drop technique
Hippurate hydrolysis positive
Esculin (+); Salicin (+)
CAMP (+)
Grows from 0.5 to 45 deg C. Cold enrichment may be used
Listeria monocytogenes
CAMP (+)
Hemolysis looks like shovel
L. monocytogenes
Used to detect beta hemolysis of L. monocytogenes
Sheep blood agar
L. monocytogenes produces blue-green colonies in this medium
Tryptose agar
Media that uses sheep blood for L. monocytogenes
McBride Listeria medium
What shall you use for contaminated specimens, which inhibits gram negative organisms
Phenylethylalcohol agar
Storage for Suspect cultures and tissues
Hold at 4 deg C up to 6 months
Subculture weekly for 1 month, and monthly thereafter
Characteristic motility of L. monocytogenes
Umbrella-shaped pattern
Virulence test for L. monocytogenes
Culture instilled in rabbit eye causes purulent conjuctivitis
Anton test
Difference of Listeria from Corynebacteria
Nonmotile, Salicin (-)
L. monocytogenes causes ___ diseases
- Gastroenteritis
- Meningitis
- Infection in pregnancy
- Granulomatosis infantiseptica
Severe in utero infection from Listeria
Disseminated abscessess and or granulomas
Granulomatosis Infantiseptica
- Slender, pleomorphic gram pos rods that form filaments;
- May be alpha hemolytic on blood agar;
- Decolorizes easily on gram stain thus may appear as gram negative
- Catalase, oxidase, indole (-)
p - H2S positive
Erysipelothrix rhusiopathiae
Disease caused by E. rhusiopathiae (aka “seal finger” and “whale finger”)
painful, pruritic, well circumscribed inflammatory skin lesion with a raised violaceous edge and central clearing
Erysipeloid
Characteristic pattern of E. rhusiopathiae in motility medium
Test tube brush pattern
Obligate aerobes
Slender, nonspore forming, acid-fast, gram positive rods (do not gram stain well)
with waxy cell wall containing mycolic acid
Mycobacterium
Stains for mycobacterium
- acid fast
- Auramine-rhodamine fluorescent stain (Truant)
Kinds of digestant for Mycobacterium
- NAC (N-acetyl-L-cysteine-NaOH) - preffered
- 2-4% NaOH
Culture media for Mycobacterium are incubated in what percent CO2
5-10%
Culture media for Mycobacterium that renders green color and is opaque
Lowenstein-Jensen (LJ)
Culture media that is good for niacin test
Lowenstein-Jensen media
Clear agar for Mycobacteria
Can see colony early
Good for antimicrobial susceptibility test
Middlebrook 7H10 or 7H11 agar
Both L-J and Middlebroom media contain ___
which enhances human strain of M. tuberculosis
Glycerol
Identification of mycobacteria is based on ___
pigment production, growth rate, and biochemical reactions
Best test for M. tuberculosis
Niacin test
M. tuberculosis is positive in 10 - 20 days using this test.
Runyon groups II and III are positive in 5 days
Hydrolysis releases oleic acid, which turns neutral red (indicator) from amber to pink
Enzymatic hydrolysis of Tween 80 (polyethylene derivative of sorbitan mono-oleate)
M. tuberculosis (+)
M. bovis (-)
M. kansasi (+ control)
Nitrate redution
Almost all mycobacteria are
catalase positive
Incubation of mycobacteria
69 deg C for 30 mins
After incubation of mycobacteria, M. tuberculosis and M. bovis are catalase ____
negative
The loss or reduction of catalase activity pf M. tuberculosis and M. bovis indicates resistance to
Isoniazid (INH)
What medium is used in which the growth of M. fortuitum complex is positive
MacConkey medium
Colonial morphology: Rough, wrinkled, dry, elevated, buff-colored (cauliflower-like) at 35 - 37 deg C
M. tuberculosis
How many weeks does M. tuberculosis grow
2-3 weeks
Niacin (+) yellow color
Tween 80 hydrolyzed in 10 - 20 days (pink color)
Nitrate (+)
Catalase ata 68 deg C (-)
No growth on MacConkey medium
Arylsulfatase (-)
M. tuberculosis
M. tuberculosis is easily killed by
Moist heat
Boiling for 10 mins
Pasteurization
Steam under pressure (autoclave)
Associated with skin lesions in tropical and subtropical areas (Buruli ulcers in Africa)
M. ulcerans
Requires several weeks of incubation at 32 deg C
M. ulcerans (6-9 weeks)
Niacin (-)
Nitrate reduction (-)
Tween 80 (-)
M. ulcerans
Photochromogens
M. marinum
M. asiaticum
M. simiae
M. kansasii
Scotochromogens
M. scrofulaceum
M. szulgai
M. gordonae (“tap water scrotochromogen”)
M. flavescens
Non-photochromogens
M. avium-intracellulare
M. malmoense
M. haemophilum
M. terrae-trivale
M. xenopi
Rapid growers
M. phlei
M. smegmatis
M. fortuitum-chelonae complex
Most common non-TB mycobacterial infection
Mycobacterium avium complex
Very rare cause of pulmonary disease in non-HIV
Causes disseminated disease in HIV/AIDS with low CD4 count (<50)
M. avium complex
Hansen’s Bacillus
Mycobacterium leprae
obligate intracellular organism; reservoir is armadillos
M. leprae
Causes Hansen’s disease (leprosy)
M. leprae
Macrophages containing acid-fast bacilli
Lepra cells
Milder form of leprosy;
strong cell-mediated TH1 response contains infections;
patches of hypopigmented skin with loss of sensation over affected area;
(+) granuloma
Tuberculoid leprosy
Severe disease;
depressed cell-mediated immunity;
diffuse skin lesions with often deformed, thickened skin ;
(-) granuloma
Lepromatous leprosy
Skin test for leprosy using a sterile extract from lepromatous nodules
Lepromin test
Early or Fernandez reaction
24 - 48 hours
Late or Mitsuda reaction
3 - 4 weeks
Thin, branching and beaded modified acid fast positive rods
Nocardia
Obligate Aerobe
Partially acid-fast
delicate mycelium
Fragmenting into bacillary forms
Catalase (+) and Urease (+)
Nocardia
Inhalation of Nocardia causes ___, which presents as a subacute to chronic pulmonary infection that may disseminate to other organs, usually the brain or skin, not transmitted from person to person
Nocardosis
Tissue picture:
Gram (+), not stained with H&E or with special fungus stains
Partially acid fast with Kinyoun stain
Nocardia
Gram neg
coffee bean shaped diplococci with adjacent sides flattened;
non-motile
Fastidious
Aerobic and capnophilic
Optimal growth in a moist environment
oxidase and catalase (+)
Neisseria
Oxidase and catalase (-) neisseria
N. elongata
-Never considered as normal flora
-Transmitted via sexual contact; infected mother to newborn during delivery
-GND, intracellular and or extracellular within polymorphonuclear neutrophils
N. gonorrhoeae
Principal virulence factor of N. gonorrhoeae
Pili
Function in adhesion of gonococci within colonies and in attachement of gonococci to host cell receptors
Opa proteins
Prophylaxis for Gonococcal ophthalmia neonatorum
1% silver nitrate solution (Crede’s method),
tetracyclin or erythromycin ointment
Neisseria spp that colonizes nasopharynx of healthy people
N. meningitidis
Outer membrane of N. meningitidis consists of ____ and ___ that play major roles in organism virulence
proteins and LPS
More severe pathology of N. meningitidis
Fulminant meningococcemia
Most common complication of meningococcemia
Meningitis
DIC and circulatory collapse (N. meningitidis)
Waterhouse-Friedrichsen Syndrome
Prone to recurrent Neisseria meningitidis infection (usu meningitis)
Patients with C5 - C9 deficiency
Rarely causes disease
Important because it grows in selective media for gonococci and meningococci from clinical specimens;
-can be cultured from the nasop
N. lactamica
Sometimes resembles N. gonorrhoeae because of its morphology and positive hydroxyprolyl aminopeptidase reaction
N. cinera
Gonococci and meningococci should be incubated in cultures in ____
5-10% CO2 and 50-70% humidity
Encourages growth of broth Haemophilus and Neisseria
Chocolate agar
enriched chocolate agar with vancomycin, colistin, nystatin (VCN) inhibitor
Thayer-Martin medium
Antibiotic that inhibits G+ organisms
Vancomycin
antibiotic that inhibits gram-neg rods
Colistin
Antibiotic that inhibits yeasts
Nystatin
Culture media for gonococci and meningococci that includes trimethoprim lactate to discourage spreading of Proteus
Modified Thayer-Martin (MTM agar)
Culture media for gonococci and meningococci that contains anisomycin instead of nystatin
Martin-Lewis Agar
ulture media for gonococci and meningococci that contains amphotericin B instead of nystatin
New York City Agar
Reagent of oxidase
1% tetramethyl-p-phenylenediamine dihydrochloride
___ in chrome loop can give a false positive reaction
Iron
4 Genera that are oxidase positive
Neisseria
Aeromonas
Moraxella
Pseudomonas
Percent concentration of H2O2 of Superoxol test
20 - 30%
IF the culture is still positive after patient is treated with penicillin, test isolate for ___ (neisseria)
Beta lactamase
Some strains of neisseria are resistant to penicillin because they produce penicillinase are called ____
Penicillinase Producing N. gonorrhoeae (PPNG)
Test used for direct detection of N. gonorrhoeae in genitourinary specimens (preferred tests for these specimens)
Nucleic Acid Amplification Tests (NAAT)
Member of the normal microbiota in 40 - 50% of healthy school children
Moraxella catarrhalis
-Encapsulated and with pili that serve as adhesins
-can be differentiated from Neisseria by its lack of carbohydrate fermentation and by its production of DNase
-produces butyrate esterase, which forms the basis of rapid fluorometeric tests for identification
Moraxella catarrhalis
Most strains of this microorganism from clinically significant infections produce ___ but are still usually susceptible to cephalosporins, trimethoprim-sulfamethoxazole and beta-lactamase inhibitor combinaations
Beta-lactamase
-Gram negative rods that are either motile with peritrichous flagella or non-motile
-Facultative anaerobes that grow well on artificial media
-Catalase (+)
-Oxidase (-)
-Reduce nitrate to nitrite
-Fermenters (produce acid in presence or absence of oxygen)
-All are glucose fermenters with or without gas production
Enterobacteriaceae
Contains lactose permease and beta galactosidase
Lactose fermenters
Contains beta galactosidase only
Late lactose fermenters
Contains no lactose permease and beta galactosidase
Non-lactose fermenters
Somatic antigen. found in the cell wall
-have lipopolysaccharide, heat stable
-used for serological grouping of Salmonella and Shigella
O antigen
-Flagellar antigens
-Located in the flagella
-Heat labile, proteins
-Used to serotype Salmonella
H antigen
-Capsular antigen
-Polysaccharide, heat labile, may mask O antigen
-Removed by heating
-Role in preventing phagocytosis
-Increasing virulence
-Vi antigen is K antigen produced by S. typhi
K antigen
Colon bacillus
Escherichia coli
EMB Green metallic sheen
E. coli
IMVC: ++–
E. coli
Diseases caused by E. coli
Diarrhea
UTI
Neonata meningitis
Gram neg sepsis
“DUNG”
Organisms that causes neonatal meningitis
Group B strep
E. coli
L. monocytogenes
Also known as Shiga toxin - producing STEC or verotxin-producing (VTEC)
Enterohemorrhagic (EHEC)
-causes diarrhea, hemorrhagic colitis, HUS
-undercooked meat, raw milk, apple cider
EHEC
Most common isolate of group and pathogen most often isolated from bloody stools
E. coli O157:H7
Culture media for EHEC
SMAC
-Traveler’s diarrhea (aka Montezuma’s revenge, Turista)
-diarrhea in infants
-transmitted by contaminated food or water
ETEC
-Bloody diarrhea, dysentery-like
-usually in young children in areas of poor sanitation
-causes invasiveness
EIEC
-diarrhea in infants (p=pediatric)
-major pathogen in infants in developing countries
-transmitted in formula and food contaminated with fecal material
-adherence - attachment
-watery diarrhea with mucus
EPEC
-Diarrhea in developing countries
-Chronic diarrhea in HIV-infected patients
-Nosocomial and community acquired
-most labs can’t detect
EAEC
Complicates 10% EHEC cases; usually occurs 5-7 days after diarrhea
TRIAD:
1. Hemolytic anemia
2. Acute renal failure
3. Thrombocytopenia
HUS
-Intestinal flora
-Have large polysaccharide capsules; nonmotile
-colonies are usually mucoid in appearance
-lysine decarboxylase (+) and citrate (+)
-causes infection among patients with impaired host defenses (alcoholics, diabetics, with comorbidites)
Klebsiella
Common ventilator-associated pneumonia and is also associated with alcoholism (pneumococcus is still more common) and aspiration
-Lobar pneumonia with a high incidence of abscesses and thick, bloody (dark red or “currant jelly”) sputum
Klebsiella pneumonia
Only indole positive Klebsiella
K. oxytoca
2 most common Enterobacter spp
E. aerogenes, E. cloacae
-Motile, almost the same IMVIC reaction as Klebsiella
-citrate and ornithine decarboxyolase (+)
- cause opportunistic and nosocomial infections
-resistant to many antibiotics
-extended-spectrum beta-lactamases
-resistance to most beta-lactams; penicillins, cephalosphorins, aztreonam
Enterobacter
-Flagellated and motile
-Encapsulated
-Produce H2S
Salmonella
In ____, Salmonella osteomyelitis is a serious and recurring problem
sickle cell patients
Typhoid enteric fever is caused mainly by ___
S. typhi
Disease and causative agent of a characteristic “rose spots” rash that may appear on the trunk of the patient in the second to third weeks.
-classically presents with pulse-temperature dissociation
-after recovery, 3% of px become carriers; the organism may be retained in the gallbladder and biliary passages
Enteric fever caused by S. typi or S. paratyphi
-nonmotile, fragile organism; closely relate to E. coli
-causative agent of shigellosis
Shigella
Most communicable of bacterial diarrhea (<200 bacilli needed to infect a person)
Shigellosis
Produces swarming on agar
Burnt chocolate odor on culture
Proteus
Most common proteus spp.
Indole (+)
TSI: K/A
P. mirabilis
-Indole (+)
-TSI: A/A because of sucrose fermentation
P. vulgaris
Struvite kidney stones then to urinary tract obstruction
Proteus
Mainly nosocomial infections
UTI
Wound infections
Morganella morganii
Slow lactose fermenters
Citrobacter, Serratia marcescens
Can be found in normal GI flora
Gram negative sepsis (with other GN bugs)
Citrobacter
Found in domesticated animals
Yersinia enterocolitica
Mimic Crohn’s disease or appendicitis
Yersinia enterocolitica
Cause of bubonic plague “black death”
Yersinia pestis
Humans get bubonic plague from rat flea bites of ____
Xenopsylla cheopis
Chief reservoir are reptiles and freshwater fish
Edwardsiella tarda
Causes UTI and diarrhea
Providencia
Only oxidase positive enterobacteriaceae
Plesiomonas shigelloides
Pleomorphic gram negative rods in singles, pairs, short chains, or long filaments
Grows on SBA and CHOC, most grow on MAC
Plesiomonas shigelloides
Most common nonfermenter
Pseudomonas aeruginosa
Gram neg, motile rods with single polar flagella
P. aeruginosa
beta hemolytic, obligate aerobe that grows readily on many types of culture media, sometimes producing a sweet or grape-like or corn taco-like odor
P. aeruginosa
Nonfluorescing bluish pigment; often produced by P. aeruginosa
Pyocyanin
Fluorescent yellow pigment
Fluorescein
Fluorescent green pigment
Pyoverdin
Dark red pigment
Pyorubin
Pyomelanin
Black pigment
Oxidizes glucose, citrate (+), oxidase (+), catalase (+)
P. aeruginosa
gives rise to blue-green pus
P. aeruginosa
Meningitis when introduced by lumbar puncture or during neurosurgical procedure
P. aeruginosa
Chronic pneumonia in cystic fibrosis patients
P. aeruginosa
An exopolysaccharide is responsible for the mucoid colonies seen in cultures from cytic fibrosis patients with chronic pneumonia
Alginate (P. aeruginosa)
“Swimmer’s ear”
invasive, malignant otitis externa in patients with diabetes
Mild otitis externa (P. aeruginosa)
Causes eye infection after surgery or surgical procedures
P. aeruginosa
P. aeruginosa Causes fatal sepsis in infants or debilitated persons; hemorrhagic necrosis of the skin in which the lesions are called
ecthyma gangrenosum
P. aeruginosa causes ___, associated with poorly chlorinated hot tubs and swimming pools
folliculitis
P. aeruginosa causes ___ in IV drug users
Osteomyelitis
