BISC 303 lab exam

Describe Kohler illumination

1. Fully rack up condenser

2. Focus on specimen with 10X

3. Close field diaphragm

4. Lower condenser until in optimum focus

5. Center field of view with centering screws

6. Open field diaphragm until hexagon just clears field of view

Describe Gram stain procedure

1. Heat slide and make a well

2. Prepare smear and air dry

3. Heat fix

4. Fill well with Crystal Violet, wait 60 seconds, rinse with water, dab

5. Fill well with iodine, wait 60 seconds, rinse, dab

6. Decolourize with ethanol for 10 seconds, rinse with water and dab

7. Stain with safranin for 10-20 seconds, rinse and blot

8. Let stain air dry

9. Observe under oil immersion

Describe KOH test

1. Place one drop 3% KOH on a slide

2. Mix one loopful of cells from plate into KOH

3. Raise loop above slide to see mucoid thread

What does a Gram positive cell look like?

Purple

What does a Gram negative cell look like?

Pink

What is a selective medium? Give an example

Incorporates 1+ reagents which inhibit the growth of certain groups of bacteria and having a minimal effect upon the growth rate of others

Ex. Conc of crystal violet

What is a differential medium? Give an example

Incorporates reagents/chemicals that permit the observer to differentiate between types of bacteria after the latter have grown, by means of colour rxn or characteristic colony morphology

Ex. blood agar

What is an enriched medium? Give an example

Made by adding 1+ nutritional supplements to a medium thereby permitting a fastidious organism to grow

Ex. chocolate medium

What are the different flagella arrangements?

Polar/monotrichous: single flagellum at one pole

Lophotrichous: tuft of flagella at one pole

Amphitrichous: flagella at both poles

Peritrichous: flagella all over

How can you see flagella through a microscope?

Layer it with a mordant to increase diameter

What affects the motility of organisms?

Temperature, due to % moisture content in the medium and the air above it

What does oxidase test conclude?

To see if the enzyme oxidase is present (useful for G-). Aerobic (and facultative in air) use oxidative phosphorylation where ETC is oxygen. Oxidase is the final enzyme which passes e- to O2

What is a + result for an oxidase test?

If paper disc turns purple to black, oxidase is present and bacteria uses oxidative phosphorylation

What is a - result for an oxidase test?

No colour change

What does catalase test conclude?

The enzyme catalase removes toxic hydrogen peroxide during oxidation-reduction reactions coupled with oxygen in respiratory metabolism

What is a + result for a catalase test?

Bubbles when you add bacteria to H2O2, showing catalase being broken down

What is a - result for a catalase test?

No bubbles between bacteria and H2O2

What does the gelatinase test conclude?

If gelatinase is present, which catalyzes hydrolysis of gelatin as a source of C and energy

What is a + result for a gelatinase test?

If the broth stays liquid, showing gelatin was broken down

What is a - result for a gelatinase test?

If broth solidifies, gelatin is present but no gelatinase

What does the amylase-starch hydrolysis test conclude?

To test for starch hydrolysis

What is a + result for amylase test?

A clear zone in the middle of the agar, showing it hydrolyzed the starch

What is a - result for amylase test?

The colour stays blue/black, so no starch was hydrolyzed

What does the indole test conclude?

To see if the amino acid tryptophan is broken down, which produces indole

What is a + result in the indole test?

Upper alcohol portion turns red, indicating indole is present

What is a - result in the indole test?

No colour change in the upper alcohol portion, showing no indole present

What does the Voges-Proskauer test conclude?

To look if the organism uses the butanediol pathway through looking at one of the intermediates, acetoin

What is a + result for the VP test?

Formation of a pink to red colour indicates acetoin, so it uses the butanediol pathway for fermentation

What is a - result for the VP test?

No colour change, so no acetoin present and organism does not use the butanediol pathway for fermentation

What does the methyl red test conclude?

It looks at mixed acid fermentation,, which produces acid that drops the pH

What is a + result for methyl red test?

If it turns red, pH < 5 and mixed acid fermentation occured

What is a - result for methyl red test?

If it turns yellow, pH > 5 and does not use mixed acid fermentation

What does the citrate test conclude?

If Enterobacteriaceae use citrate as sole source of C and energy

What is a + result for citrate test?

If it turns to blue (and sometimes yellow), ammonia was formed and increased the pH through using citrate

What is a - result for citrate test?

If there is no colour change (stays green), pH did not change and citrate was not used. If there are heavy inoculums, yellow may appear which is also - result

What does the phenol red test conclude?

The fermentation of carbohydrates. The organism may produce acid and/or gas of neither

What is a + result for phenol red test?

If the tube turns yellow, pH < 6.8. If it turns red, pH > 8.4. If there are bubbles in the vial, gas was produced

What is a - result for phenol red test?

No colour change and no bubbles in vial

What does the nitrate test conclude?

Nitrate is TEA for anaerobic respiration. Nitrate is reduced to nitrite (NO3->NO2-). Some bacteria reduce the toxic nitrite further to NO, N2O, or N2 gas. This respiration is dissimilatory as N is a waste product

NO3- -> NO2- -> N2, NO, N2O, or NH3

What is a ++ result for nitrate test?

No colour change after zinc is added as nitrite was further reduced to NO, N2O, or N2

What is a + result for nitrate test?

The development of a red, purple, or maroon colour without adding zinc, showing nitrate was reduced to nitrite only

What is a - result for nitrate test?

Colour change to red, maroon, or purple after zinc is added, showing nitrate was not previously reduced to nitrite

What does the TSI test conclude?

To identify G- organisms based on their ability to ferment glucose, lactose, and sucrose as well as produce hydrogen sulfide gas

What is a K/A result for TSI test?

Alkaline slant/acid butt = yellow/red

Glucose only was fermented

What is a A/A result for TSI test?

Acid slant/acid butt = red/red

Glucose + sucrose, glucose + lactose, or glucose + sucrose + lactose

What do bubbles and cracks mean in a TSI test?

Gas formation

What does black precipitate mean in a TSI test?

Hydrogen sulfide gas was produced

What does the API test conclude? How do you make it anaerobic?

To differentiate between G- isolates using percentages of results. When required, organisms are forced to ferment by creating anaerobic conditions with mineral oil

What is the provincial waste standard for fecal coliforms in water?

200 coliforms/100mL

Why would you want to do ribosomal sequence analysis?

To identify and classify bacteria

How do you do ribosome sequencing analysis?

Use PCR to amplify a portion of 16S rRNA using a universal bacterial primer. Success of PCR is determined by agarose gel electrophoresis. Then, purified PCR product will be sent off for sequencing

How does the BLASTN tool work?

It compares sequencing results from PCR with sequences in a database that share similarities (including Bacterial and Archaeal small-subunit 16S rRNA gene sequences

Why should you open R2A plates in biosafety cabinet (BSC)?

R2A agar has a low carbon to volume ratio and was cultured at a low temp to allow heterotrophs to grow. These bacteria are uncharacterized, so could be dangerous

Why is the polymerase chain reaction (PCR) technique sensitive?

It allows the amplification of trace amounts of template DNA, so is very prone to contaminants. Trace amounts of contaminants can lead to false results. As well, nucleases from hands can destroy template DNA. Must handle with gloves, and clean materials

Why is it important to keep reagents containing enzymes and other biological ingredients on ice when using molecular techniques?

It prevents degradation or unintended reactions from taking place

What is the process of PCR?

Heat denatures DNA, which then is extended using the primers. Then refrigerated overnight

What is the usefulness of agarose gel electrophoresis?

It tests the success of PCR. The universal bacterial primers should result in an approximately 960 bp PCR product on gel (lower section). Size of PCR product is established by comparison to a ladder of DNA bands of known size

What are the precautions in using agarose gel electrophoresis?

The dyes used to stain DNA are potential carcinogens

Why might PCR not work?

Human error (contamination or inaccurate pipetting), colony was archaea, mold, or yeast which won't work contain the universal primer sequence in genome. Even some bacteria might not work as it is unclear how universal this primer sequence is

How do you test for horizontal transfer of antibiotic resistance through strains?

Conjugation, through using a spread plate with both antibiotics and determining growth. The donor strain is Hfr so it has the tra gene on its chromosome and has been transformed into a plasmid which carries the resistance. The recipient strain does not have a plasmid but is resistant to another strain

What assumption is the plate count technique based on?

The assumption that each viable cell in an agar medium has the potential to develop into a colony, as well as each colony arising from a single cell

What is the process for the plate count technique?

1. Dilute specimen

2. Plate dilution by spread plate technique

3. Incubation of plated specimens

4. Count colonies

What is optical density?

A spectrophotometer measures turbidity of liquid culture. Bacteria do not absorb light

When should you use direct counting of organisms?

Can be used to count viable cells, as dead cells stain blue while living ones do not. You count them on a hemocytometer with known volume and dilution factor

What does ELISA test for?

In immunology to detect antibodies in antigens

Why do we wash the wells for ELISA?

To remove any unbound antigen from the wells

What is the purpose of the blocking agent for ELISA?

To coat any exposed plastic with a non-specific protein so antibodies don't get stuck to the plastic (often BSA)

What is the purpose of examining bacteriophage virulents?

To determine number of PFU (plaque forming units) in the lysate

What is the Kirby-Bauer disk method?

To determine the susceptibility of an organism to an antibiotic. The effectiveness of the antibiotic is shown as a growth inhibition zone. The diameter of this may be measured in order to compare relative efficacy of microbial agents. This constitutes an antibiogram

What is the minimum inhibitory concentration (MIC) method?

The lowest concentration at which growth of a specific organism with an antibiotic will be completely inhibited

What is the agglutination method?

Identification and titer of both antibodies and antigens (whole cells). Antiserum is mixed with homologous antigen and incubated to observe agglutination (clumping) of cells. The antibody-antigen complexes will only form and fall to the bottom when the antibody titre is sufficient

What is the immunodiffusion-ouchterlony plate?

Precipitation of antibody antigen complexes in solution can be used to measure antigen/antibody concentrations or study similarities between different antigens. When an antibody and antigen are placed on adjacent areas of a gel, they diffuse into each other. There will be an opaque band where the precipitation reaction occurs readily and the antibodies are homologous to the antigens in serum. The distance between the line and antigen well decreases with decreasing antigen concentration and can be used to measure the titers of the antigen and antibody. It can be determined if various antigens have common antigenic components