Microbio Lab Exam 2

Enteric bacteria

gram-negative proteobacteria, usually rods, chemoorganoheterotrphic, facultative anaerobes, capable of aerobic respiration of many compounds, many respire anaerobically as well. They will ferment glucose/other sugars, produce catalase enzyme and do not produce cytochrome c oxidase

KIA, Kligler Iron Agar

only for gram-neg bacteria, tests or lactose/glucose fermentation. Lact: Pos = yellow slant, glucose: Pos= yellow butt of tube ± has production, also tests for production of hydrogen sulfide: pos= black precipitate on slant

MR, Methyl Red

used to identify end product of glucose fermentation (acid, acid and gas, or neutral products), red = pos for acid ph<4.4, yellow = neg for acid pH>6.2, orange= equivocal reaction

Simmons Citrate

test fort he use of citrate as a carbon/energy source, utilization of citrate pos= bright blue broth

Eosin-Methylene blue (EMB)

only allows for growth of gram negative bacteria, identifies lactose fermenting bacteria. Lactose fermenting: pos= colored colonies (black/green or pink/purple) neg= white/clear colonies

Phenylalanine

tests for deamination of phenylalanine, pos = dark green after addition of FeCl3

Motility indole ornithine (MIO)

Tests for motility: pos = cloudiness, tests for indole production: pos = red ring after adding kovacs reagent (due to breakdown of tryptophan), test for ability to decarboxylate ornithine: pos = grey/blue/purple color in lower ¾ of tube

Lysine decarboxylase

tests for decarboxylation of lysine (also ornithine and histidine), pos = gray/blue/purple color in tube

Decarboxylase control test

must be done concurrently w/lysine decarboxylase test, all enteric should be able to grow in this control, need a yellow color to continue to look at other lysine decarb test.

API test strips

small versions of all the tests in one strip. API 20 specifically tests for enteric bacteria

Traditional tests

glucose fermentation nitrate reduction, Motility, catalase, and oxidase

Glucose fermentation

tests for glucose fermentation (acid, acid+gas, no acid or gas), pos = yellow color and or gas bubble in durham tube

Nitrate reduction test

tests aerobic respiration with nitrate as the acceptor, NO3→ N2 gas = large bubble in durham tube, if no bubble, add sulfanilamide and N-(1-napthyl)-ehylenediamine, if pink pos for NO3→NO2. If not pink add zinc, if pink/red there was no deamination of NO3

Motility test

tests for motile microbes, cloud = motile

Catalase test

tests for the enzyme catalase, strict aerobes/facultative anaerobes expected to posses catalase, pos = bubbling after adding 3-5% H2O2 to plate.

Oxidase test

Test for cytochrome c oxidase enzyme, it is a plastic strip, immediate color change indicates positive test

Genotypic Characterization of microbes

Can be done by sequence analysis of microbial DNA

Sequence Analysis of DNA

Most common: small subunit ribosomal RNA gene, matches SSU rRNA to many known species. traditionally done with pure cultures but can use PCR on complex samples. DNA (&RNA) sequences are also used for detection of microbes in a given environment (involves use of DNA probe)

Serological Methods

taking advantage of specific features that vary between microbes, based in antibody technology

Antibody technology

Using antibodies to see if microbe has the matching antigen, can be done by radial immunodiffusion, double diffusion agglutination, and enzyme immuno assays

Lactic Acid Bacteria

most important group for fermenting foods, gram-positive cocci, aerotolerant anaerobs/obligate fermenters, no cytochrome and no catalase, resistant or azide. found: associated with plant material and animal hosts. Foods: cheese, buttermilk, yogurt, sausage prod, vegetable fermentation, can be responsible for spoilage

Yeasts

ethanol-fermenting microbes, Food/drinks: bread, alcohol, kombucha

Acetic Acid bacteria

gram-negative bacteria, aerobic respiration (has functional electron transport chain), converts ethanol to acetic acid. Foods: vinegar, chocolate, and kombucha.

Differential Media (food microbes)

All purpose tween agar + NaN3, YTG agar, Sabourauds dextrose agar, and GYEC agar

All purpose tween agar (APT) + NaN3

for optimal enrichment and isolation/growth of lactic acid bacteria, has azide in it so it will select for obligate fermenters

YTG agar

All purpose, non-selective agar

Sabourauds Dextrose Agar

Favors growth of acid tolerant microbes, including many fungi

GYEC agar

For isolation of acetic acid bacteria, white medium

Indicator organisms

used as a measure of fecal contamination to asses water quality, Needs 4 properties:

1. only natural environment should be in association with feces

2. shouldn’t grow in nature outside of its natural environment

3. should survive longer than most viable pathogens but not so long that historical events are detected

4. should be easy to detect

Coliform/fecal coliform bacteria

one group within enterobacteriaceae (the coliform), closest to fulfilling the criteria of indicator organisms and used as a measure of biological contamination in water. Non-spore forming, facultative anerFobes, gram-negative rods, ferment lactose to acid/gas

Fecal coliforms

capable of lactose fermentation to acid and gas, most strains are E. coli (not all), E coli are good indicators of fecal pollution because they dont survive long in enionrment out of host and can be differentiated from other coliform by incubating on selective media at 44.5 degrees Celsius. Most are not pathogenic

MPN

most probable number, statistical method to get quantitative data on the concentration of bacteria based on presence/absence of growth. 2 stages: presumptive and confirmed, Math: MPN # x middle dilution factor (10^-3 would be 10³) = coliforms per mL in OG sample

Presumptive

dilutions of water samples added to series of LLTB tubes, pos growth recorded

Confirmed

Samples from pos presumptive test inoculated into BGLC/EC tubes, positives from those used to calculate MPN in original sample

LLTB tubes

growth and gas = positively presumptive that coliforms are in sample

BGLB tubes

growth and gas = positive for coliforms

EC broth

growth and gas = positive for fecal coliforms

Colilert Test

Predicts most probable number of coliforms and fecal coliforms in a sample, 100mL water sample divided into 51 pouches. ONPG and MUG in the pouches, these are metabolized by coliforms or E coli.

ONPG

metabolized by coliforms and turns yellow

MUG

degraded by E. coli and turns florescent under UV light

Determining # of microbes in food sample

Dilute food with saline, and create serial dilution scheme (1:100, 10^-4, 10^-5, 10^-6), plate dilutions of MAC and PCA plates. Let incubate and count plates with 30-300 colonies

PCA

plate count agar, all purpose rich complex media

MAC

MacConkey Lactose Agar, inhibits gram-positive bacteria bc of bile salts/crystal violet. Coliforms are gram-neg so should grow on this plate.

Anibiotics

chemical agents produced by microorganisms that can kill or inhibit certain other microorganisms. Sensitive = susceptible, many bacteria are resistant or various antibiotics (E. coli resistant to vancomycin), antibiotics have to pass through porins in gram-neg, or through thick peptidoglycan in gram-pos bact. Gram-pos more susceptible to anitboitcs that target peptidoglycan synth. Able to target bacteria specific components of cell function, so it does not mess with human cells

Antibiotic Disk Diffusion Assay

Small paper disk with antibiotic is placed on a lawn of bacteria, antimicrobial agent is then diffused into medium. A concentration gradient is established with a high concentration of agent near the disk and gradually declines as it goes out. If effective it will kill/inhibt growth.

Resistant

Small to no ring

Intermediate

Medium ring around disk

Susceptible/Sensitive

large ring around disk of no bacteria

Minimal Inhibitory Concentration

MIC, uses liquid broth, allows for lowest inhibitory concentration to be found, useful when determining dosage, set up: 6 tubes with YTG broth, prepare 2-fold increases in antibiotic concentration, leaving one control tube (0, 6.25, 12.5, 25, 50, 100). Then innoculate with 10 microL of bacteria, use c1v1 = c2v2 to find new concentration of antibiotic in tube