Intro to Bacteriology: Collection & Diagnostics Flashcards
Hierarch of Classification
Kingdom (Domain)
Division (Phylum)
Class
Order
Family
Genus
Species
Subspecies
“Strains”
Classification - Genus
Different species in same genus share common genetic/phenotypic features
Minor differences - different species within same genus
Classification - Species
Most basic taxonomic group
Common or similar physiologic and genetic features
Biotype and serotype - variants in the same species
Nomenclature Rules
- Names are in Latin/latinized
- First letter of genus capitalized, species is lowercase
- Italicized or underline
Phenotypic Identification
Macroscopic/microscopic traits
Nutritional/environmental requirements
Biochemical properties
Antigenic traits
Antibiotic resistance/susceptibility
Genotypic Identification
Nucleic acid traits
Gram Positive Cell Wall
Three main things + specific pathogenicity
Thick petidoglycan: cell shape, units of NAG linked to NAM
Teichoic/Lipoteichoic acid: negative charge, antigenic, role in pathogenicity (adherence), + cell division
Lipoprotein: cell transport
Gram Negative Cell Wall
Three main things + specific pathogenicity
Outer membrane: OM is for cell protection, permeability
Lipopolysaccharide (LPS): component of OM, antigenic, pathogenicity (endotoxin)
Thin peptidoglycan
Mycobacteria Cell Wall
Four main things + specific pathogenicity
Mycolic acid: cell protection, permeability
Araginogalactan: connects peptidoglycan to mycolic layer
Thin peptidoglycan
Lipoarabinomannan: cell wall integrity, antigenic, pathogenicity (immunomodulator)
Gram Stain Procedure
And bacterial appearance
Fixation
Crystal Violet
Iodine treatment
Decolorization
Counter stain safranin
Gram positive stain purple
Gram negative stain pink/red
Some are gram-variable or don’t stain
Acid Fast Stains
What for, three types of stains
High lipid and wax in bacteria cell walls don’t stain well with traditional stains
Ziehl-Neelsen: hot method
Kinyoun: cold method
Both use carbolfuschin
Fluorescent stain: auramine O for flurochrome dye
All of the above use phenol for penetration into cell wall
Endospore Stain
Basically what are spores and how are they different
What kind of bacteria form spores?
Gram positive bacteria can form spores (endospores for survival in harsh conditions)
Resistant to UV, temperature, chemicals
Capsule Stain
What do capsules have, what does it cause to normal stain?
Stains background with acid and cell using basic stain
Capsules have polysaccharides or polypeptides
Non ionic -> don’t stain with acid or basic stains
What method does bacteria use to multiply?
Binary fission
Generation time - time it takes for 1 cell -> 2 cells
Bacterial Growth Curve
4 phases
Lag phase: adapt to conditions, cells are maturing but not dividing
Log phase: cell doubling
Stationary phase: growth rate slows, nutrient depletion and more toxic products. Maximum cell number
Death phase: run out of nutrients and die
Bacteria produce more antibiotics and spores during stationary due to lack of nutrients
Antibiotics work on bacteria when they were at log phase
Heterotroph
Bacteria that inhabits human body
Require organic source of carbon
Nutritional Media Classifications
4 types
Non-inhibitory/nutrient: complex, extract of meat + soybeans
Enriched: added growth factors, blood, vitamins, enhaces growth of certain bacteria (ex. sheep’s blood)
Selective: agents that are inhibitory to some, allows growth of choice
Differential: visual metabolic characteristics, distinguish microbes growing (ex. MacConkey agar)
Sheep Blood Agar
Three types of hemolysis
Contains RBCs
Alpha hemolysis: reduces RBC hemoglovin to methemoglobin, causes green/brown color
Beta hemolysis: complete lysis of RBC, clear zone on agar, bacteria can destroy RBCs
Gamma hemolysis: lacking lysis
MacConkey Agar
Properties and visuals?
Differential properties: lactose positive produce pink colonies, lactose negative are colorless
Selective properties: G+ inhibited by bile salts and crystal violet, inhibits Proteus spp.
Best pH for Bacterial Growth
Neutral (7-7.5)
Temperature of Biological Growth
-philes and temperature ranges
Mesophilic: body temperatures (37 / 35-37 C)
Psychrophiles: cooler temperatures (4-20 C)
Thermophiles: warmer temperatures (42 C)
Atmospheric Requirements for Growth
Oxygen?
Obligate aerobes: needs oxygen, 21%
Obligate anaerobes: no oxygen
Facultative anaerobes: aeorbic and anaerobic
Capnophiles: increased CO2
Microaerophiles: reduced oxygen, increased CO2
Identification of Organism (Tests)
3 different types of tests
Biochemical enzymatic capabilites - can detect single enzymes, oxidation, fermentation, and amino acid degradation
Antigenic properties: using antibodies to detect pathogenic antigen (such as agglutination)
Resistance/susceptibility profiles: growth in certain inhibitory or toxic conditions (think zone of inhibition on a plate)
Time of Collection for Testing
How time can affect the test
Acute phase: etiological agents more likely to be detected
Before antimicrobial therapy: some organisms are killed and cannot be recovered
Time of day: first morning is best for some
Specimen Transport
Room temp: wound, body fluid, genital specimens, swabs in transport media
Fridge/frozen: specimens that are likely to contain microbial flora and viral specimens
- Neisseria meningitidis, N. gonorrhoeae, Heaemophilus influenzae are harmed by cold temps
Preservatives: urine/feces
Specimen Processing Priority
Urgent: life-threatening
Routine: no risk, potentially important though
Elective: more confirmatory than diagnostic
Specimen Processing Methods
3 ways to id bacteria
Macroscopic: consistency, volume, blood/mucus, color, clarity
Microscopic: quality of specimen, routine workup, additional testing
Culture media: use prior information to determine what kind of media is needed
Blood Specimen Cultures
Collection: cleanse and draw correct blood amount according to adult or child
Prior to antibiotics/at fever spikes, 2-4 sets within 24 hours
Anaerobic and aerobic bottles
Next: gram stain, biochem tests, subculture to media
Urine Specimen Cultures
Specimen collection: prevent contamination, refridgerated if not used after 30 minutes
Culture: calibrated loop, SBA and MAC media
Next: count colonies from SBA, idenitification
Stool Specimen Cultures
Collection: taken during acute phase, process within 72 hours
Culture: routine pathogens, selective media, immunoassays available
Next: review plates and identify
Respiratory Tract Specimen Cultures
Collection: avoid contamination, smear exam
Culture: onto media
Next: review plates and identify
Catalase Assay
“Rapid” biochemical assay
Hydrogen peroxide in human cell, organisms must rely on defense to escape H2O2 damage
Catalase breaks down H2O2: positive test is when the sample bubbles with H2O2
Coagulase
“Rapid” biochemical assay
Has two types of coagulation
Enzyme that clots fibrinogen containing plasma
Cell wall bound coagulase: acts directly on fibrinogen in plasma
Extracellular coagulase: liberated by the cells
Pyrrolidony Peptidase (PYR)
“Rapid” biochemical assay
L-pyrrolidonyl-beta-naphthylamide hydrolyzed by the peptidase
Releases free beta-naphthylamide
Positive if ppink, orange, or red within 2 minutes
Bile Solubility
“Rapid” biochemical assay
Sodium desoxylcholate lower surface tension in membrane and makes cells lyse easier
Positive if cells are lysed in 30 mins
Oxidase
“Rapid” biochemical assay
Cytochrome C oxidase: oxidizes test reagent, porsitive if purple/blue in 30 seconds
Butyrate
“Rapid” biochemical assay
Hydrolysis of bromo-chloro-indolyl butyrate and releases indoxyl
exposed to oxygen, forms indigo
Positive if blue-violet within 5 minutes
Spot Indole
“Rapid” biochemical assay
Tryptophanase
Degrades into things + indole
Positive if red dye turns blue
Urea Hydrolysis
“Rapid” biochemical assay
Urease
Forms ammonium carbonate, turns yellow alkaline media to pink
Positive if pink within 4-24 hours
MALDI-TOF
What it does
Protein characterization
Matrix of sample mix ionized by laser beam, transfers charge to analytes and generates singly charged ions. These ions are separated based on mass to charge ratio and measured.
Molecular Techniques
Pros, examples
Pros: high sensitivity, specificity, safety
Examples: hybridization, amplification, sequencing
Serologic Tests
Pros, cons
Using antibodies and antigens
Pros: detect pathogens that are hard to culture, monitoring therapy
Cons: delay in antibody production, immunocompromised patients can have lower or no antibodies, cross reactivity
