Intro to Bacteriology: Collection & Diagnostics Flashcards by avery t.

Phenotypic Identification

Macroscopic/microscopic traits
Nutritional/environmental requirements
Biochemical properties
Antigenic traits
Antibiotic resistance/susceptibility

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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

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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

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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)

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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

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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

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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

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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

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What method does bacteria use to multiply?

Binary fission
Generation time - time it takes for 1 cell -> 2 cells

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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

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Heterotroph

Bacteria that inhabits human body

Require organic source of carbon

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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)

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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

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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.

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Best pH for Bacterial Growth

Neutral (7-7.5)

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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

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 ## Footnote "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 ## Footnote "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) ## Footnote "Rapid" biochemical assay

L-pyrrolidonyl-beta-naphthylamide hydrolyzed by the peptidase *Releases free beta-naphthylamide* ## Footnote Positive if ppink, orange, or red within 2 minutes

Bile Solubility ## Footnote "Rapid" biochemical assay

**Sodium desoxylcholate** lower surface tension in membrane and makes cells lyse easier ## Footnote Positive if cells are lysed in 30 mins

Oxidase ## Footnote "Rapid" biochemical assay

**Cytochrome C oxidase:** oxidizes test reagent, porsitive if purple/blue in 30 seconds

Butyrate ## Footnote "Rapid" biochemical assay

Hydrolysis of *bromo-chloro-indolyl butyrate* and releases indoxyl exposed to oxygen, forms indigo ## Footnote Positive if blue-violet within 5 minutes

Spot Indole ## Footnote "Rapid" biochemical assay

**Tryptophanase** Degrades into things + **indole** ## Footnote Positive if red dye turns blue

Urea Hydrolysis ## Footnote "Rapid" biochemical assay

**Urease** Forms ammonium carbonate, turns yellow alkaline media to pink ## Footnote Positive if pink within 4-24 hours

MALDI-TOF ## Footnote 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 ## Footnote Pros, examples

**Pros:** high sensitivity, specificity, safety **Examples:** hybridization, amplification, sequencing

Serologic Tests ## Footnote 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