MCB3020L Final Exam Study Guide Part 1 (General)

condenser of microscope

two lenses that focus the light rays coming from light source onto the specimen

Ocular lens of microscope

the lens that you look through, fixed at 10x

stage of microscope

platform that supports the specimen and holds it in place

stand of microscope

provides framework for optical components

microscope head

structural part that supports the ocular lens

microscope objective lens

lens above the specimen

types of objective lens

Scanning lens (4x)

Low power lens (10x)

High Dry Lens (40x)

Oil immersion lens (100x)

iris diaphragm of microscope

Adjusts the amount of light that reaches the specimen.

field diaphragm of microscope

controls diameter of light entering condenser, changing contrast

direct stain

dyes the cells, but not to the external medium or surrounding tissue ex. methylene blue

negative stain

a simple stain that stains the background but leaves the bacteria unstained, letting light pass through the cells

simple stain

stains a smear with one dye, against an unstained background. determines morphology, size, and arrangement

differential stain

uses at least three chemicals on a smear.

primary stain

colors all cells ex. crystal violet

Decolorizer

gives color contrast by removing the color from one cell type ex. ethanol

counterstain

gives contrasting color from primary stain to cells that were decolorized ex. safranin

What is a Gram stain?

A differential staining method that distinguishes between two different kinds of bacterial cell walls.

What is the primary stain used in Gram staining?

Crystal violet

What is the mordant used in Gram staining?

Iodine

What is the decolorizer used in Gram staining?

Ethanol

What is the counterstain used in Gram staining?

Safranin

What is a capsule stain?

A negative stain used to view capsules.

What does the primary stain do in a capsule stain?

It stains both the capsule and the bacteria.

What is the decolorizer used in capsule staining?

20% copper sulfate.

What is the purpose of the decolorizer in capsule staining?

It removes the stain from the capsule.

Gram positive bacteria tests

Blood agar test

Catalase test

MSA test

Gram negative bacteria tests

MacConkey Agar test

Eosin methylene blue levin (EMB) test

Salmonella-Shigella agar (SS) test

TSI agar test

Citrate test

What is the Blood Agar Test used for?

Detecting hemolytic ability of gram positive cocci.

What type of hemolysis is indicated by no change in the surrounding medium?

Non/gamma-hemolytic.

What does alpha-hemolytic indicate?

Incomplete lysis of blood cells, resulting in a green aura around colonies.

What causes the green aura in alpha-hemolytic bacteria?

Unused heme iron released from hemoglobin being oxidized.

Which organisms typically exhibit alpha-hemolysis?

Organisms in mouth flora.

What does beta-hemolytic indicate?

Complete lysis of blood cells, resulting in a clear halo.

What type of bacteria are typically beta-hemolytic?

Pathogenic bacteria.

What does the catalase test identify?

It identifies between staphylococci and streptococci.

What is added to the sample in a catalase test?

0.3% hydrogen peroxide.

What indicates a positive result in a catalase test?

Bubbles will form if the sample has catalase.

catalase

converts hydrogen peroxide to water and oxygen

staphylcocci have catalase while streptococci do not

What does MSA stand for?

Mannitol salt agar

What type of media is Mannitol salt agar?

Selective and differential media

What is the concentration of NaCl in Mannitol salt agar?

7.5% NaCl

What sugar is present in Mannitol salt agar?

Mannitol

What pH indicator is used in Mannitol salt agar?

Phenol red

What does Mannitol salt agar test for?

An organism's ability to grow in a high salt environment and ferment mannitol

Purpose of mannitol in MSA agar?

if the organism can ferment it, acid will be produced, which will turn the colonies yellow. If not, the medium won't change color.

What type of media is MacConkey agar?

Selective and differential media

What substances in MacConkey agar select for gram-negative bacteria and inhibit gram positive bacteria?

Bile salts and crystal violet

What does lactose in MacConkey agar test for?

It tests for gram-negative bacteria that can ferment lactose (coliforms) or can't ferment lactose (paracolons)

What color does neutral red turn at a pH less than 7 in MacConkey agar?

Pink

EMB agar

- eosin methylene blue levine

- selective and differential

- differentiates enteric lactose fermentors and non-lactose fermentors

eosin and methylene blue

inhibits gram positive growth

Positive results for EMB agar test

deep purple or blue-black with a green metallic sheen due to strong acid production due to fermentation

negative result for an EMB agar test

colorless colonies or a light pink or purple

What does SS agar stand for?

Salmonella-Shigella agar

What is the purpose of SS agar?

It is a selective/differential medium to identify salmonella and shigella species from other bacteria.

What components in SS agar select against gram positive and some gram negative bacteria?

Bile salts and brilliant green dye

What does neutral red in SS agar indicate?

It turns red from acids produced by lactose fermentation, but remains invisible when there is no fermentation

What does lactose in SS agar test for?

It tests for coliforms (lactose fermentors) and paracolons (non-lactose fermentors).

what is the sodium thiosulfate and iron in SS medium for?

tests for hydrogen sulfide gas production, where the thiosulfate is used as a source, while ferric citrate is used an indicator which turns colonies black when hydrogen sulfide gas production occurs

What does TSI stand for in the TSI agar test?

Triple Sugar Iron

What does the TSI agar test differentiate bacteria based on?

Glucose/lactose/sucrose fermentation, hydrogen sulfide production, and general gas production.

What are the sugar concentrations in TSI agar?

0.1% glucose, 1% lactose, 1% sucrose.

What happens when glucose is depleted in TSI agar?

Cells will break down lactose and sucrose if they can; if not, they will use proteins and turn the agar pink.

What color does the agar turn when sugar fermentation occurs in TSI agar?

Yellow.

What compounds in TSI agar test for hydrogen sulfide gas production?

Sodium thiosulfate and ferrous sulfate.

What indicates that bacteria produce hydrogen sulfide gas in TSI agar?

The formation of a black precipitate.

What does the presence of bubbles in the agar indicate in the TSI agar test?

The organism produces gas.

how is TSI poured

agar is poured into a test tube as a slant with an anaerobic but and an aerobic slant

pH indicator in TSI agar

phenol red, which turns yellow at an acidic pH (from glucose fermentation) or pink at an alkaline pH (from protein metabolism)

what happens in TSI if the organism can metabolize only glucose?

if only glucose fermentation is possible, the butt will remain yellow but the slant will turn pink

Citrate test

tests for ability to use citrate as a sole carbon source.

contains sodium citrate.

positive result of citrate test

Organism possess citrase, which will metabolize citrate and produce Carbon dioxide, turning the media alkaline

What does bromothymol blue do in citrate agar?

if the organism can metabolize citrate, the pH of the media will go up and turn blue, if not, the agar remains green

KOH test

confirms Gram stain based on whether 3% KOH can lyse bacterial cell wall and release DNA

- suspension becomes viscouse and forms string: gram negative

- suspension stays thin and no string: gram positive

What can happen to cultures older than 3 days in a KOH test?

They can lyse spontaneously even if they aren't gram negative.

How does a small inoculum affect the KOH test?

It makes it hard to see viscosity if the cells lyse.

What effect does a big inoculum have on the KOH test solution?

It can make the solution appear viscous even if it hasn't lysed.

What happens if KOH test results aren't read within 1 minute?

The alkali will start to lyse gram positive bacteria walls and show a false negative.

First step of gram stain

primary stain: crystal violet

what happens if you skip first step of gram stain?

all the cells will be the same color as the counter stain

second step of gram stain

mordant: iodine

fuses with crystal violet and attaches it negative charged groups of cell wal

what happens if you skip second step of gram stain

dye won't stick to cell wall and will wash away easily

third step of gram stain

decolorize with ethanol (differential step to see whether Gram-positive/ negative by either trapping the violet or rinsing it away)

what happens if you skip this step

all cells will stay purple and the counterstain won't work

fourth step of gram stain

safranin counterstain (stain Gram-negative bacteria pink)

heat fixing procedure

Let inoculated slide COMPLETELY air dry.

Attach a clothespin to the slide, pass the inoculated slide through the inner flame of a Bunsen burner flame 3 times. Cool.

why is heat fixing bacteria done

glues cell to a slide by denaturing cellular proteins, which cause the amino acid side chains to stick to the slide.

chemically defined media

exact chemical composition is known

complex media

unknown exact composition, made up of complex substances ex. TSA or TSB

enriched media

complex media that has additions to allow fastidious organisms to grow, ex. blood agar

selective media

suppress unwanted microbes and encourage desired microbes

differential media

contains components that allows bacteria to be distinguished by change in appearances.

selective and differential media

selective growth and characteristic changes of those that grow

zone of inhibition

The zone where bacteria can't grow around a given antibiotic.

found by measuring the diameter of the clearing

What does the Kirby-Bauer test measure?

Zone of inhibition to determine antibiotic susceptibility based on a standardized range.

Bactericidal antibiotics

antibiotics that kill bacteria

Bacteriostatic antibiotics

Antibiotics that do not actually kill bacteria but rather inhibit their growth

how to prescribe the best antibiotic by analyzing susceptibility.

1. perform a kirby-bauer test or serial dilutions

2. for kirby-bauer tests: check if the zone of inhibition is in the standard range of susceptibility for that antibiotic

3. for serial dilutions: analyze the MBC and MIC

df

dilution factor

how to calculate dilution factor

volume of sample added/total volume (sample volume + blank volume)