Microbiology Lab Exam Semester 2

What kind of microscope are we using?

Bright field compound light microscope

What power are the ocular lenses?

10X

Name the four objective lens and their powers

* 4x Scanning
* 10x Low power
* 40x High dry power
* 100x oil immersion

Name all pieces of the microscope and describe them if necessary

* Ocular lens (eye piece)
* Diopeter (adjusts left eye)


* Objective lens
* Stage clips
* Aperture: lets light in
* condesner: controls light
* illuminator: light source
* head (big part that holds eye pieces)
* arm
* mechanical stage
* coarse and fine knobs
* stage controls
* base
* light switch

How do you calculate total magnification?

ocular times objective power

What is immersion oil for?

Stops light scattering

List and describe the common bacterial shapes

* cocci (round)
* diplococci (two)
* Streptococci (chain)
* Staphylococci (cluster)
* tetrad (four)
* cocobacillus
* bacillus
* diplobacilli
* streptobacilli

Why do we do aseptic technique?

Get pure, uncontaminated cultures

What’s the difference between agar and broths?

* Agar: solid media, in slants or plates
* Broths: liquid media

Define direct staining vs indirect staining

* Direct staining: Positive stain attaches to negative bacteria
* Indirect staining: Negative dyes stain the background

What are the 3 reasons we stain bacterial cells?

* Improve contrast between cells and slide
* To help identify bacteria
* observe cell structures

What is a negative stain used for? How do they work? What is unique about this stain?

To see size and shape of bacteria. They stain the background. You don’t heat fix the slide, therefore the bacteria aren’t killed

Why do we do smear prep?

It evenly spreads the bacteria on the slide. They can’t be washed off or get distorted

Give the basic procedure of smear prep

* put loop of water on the slide
* put loop of bacteria on slide
* Wait for it to dry
* Heat fix

What reagents are used for the negative staining?

* nigrosin

What is the purpose of gram stain, explain how it works? What does it look like?

* Divides bacteria into gram positive and gram negative
* Gram positive retain the purple, crystal violet, while gram negative loses it and takes the on the safranin pink
* Gram negative is pink, gram positive is purple

What dyes do you use for gram stain?

* Crystal violet
* Iodine
* Ethanol
* Safranin

Describe the purpose and appearance of capsule slides

* Show capsule.
* Capsules appear as unstained, white halos around red purple cells.

What is the acid fast stain used for? Appearance?

* Differentiates bacteria with and without mycolic acid in their cell walls.


* Acid fast bacteria are pink rods
* Non-acid fast bacteria are blue cocci

Why do you have to use steam heat in the acid fast stain?

The heat partially liquifies the waxy cell layers. Acid fast cells will let the first stain in, but won’t let it out or get a new one in

What dyes do you use in acid fast stains?

* Carbolfuchsin
* Acid-alcohol
* methylene blue

What is the purpose of the endospore stain? What does it look like?

Endospore stain is used to see spores

* Vegetative are pink
* spores are green

Why do we have to use heat for endospore

The heat helps drive the primary stain through the many layers of the endospore

What dyes are used for endospore stain

* Malachite green
* safranin

What is the difference between brownian and true motility?

* Brownian motility: water molecules move the cells
* True motility: self propulsion with flagella

Explain how you do hanging drop slide? Why do you do it this way?

You place a slip cover over live cells, held up by dabs of vaseline

* This allows cells to still be alive

What does epifluorescence microscopy used for?

Visualize specimens that fluoresce, that means emit light of one color

What is Scanning electron microscopy used for? How?

Sends narrow beam of electrons back and forth over specimen

Used for viewing super small specimens that must be dried

What stains do you use in epifluorescence, give both methods? What do they mean?

* Live/dead: Dead cells with damaged membrane are red from PI stain, live cells are green from syto9
* Acridine orange: cells stain green to orange. DNA stain for all cells.

Define sterilization

The process of removing all living cells, spores, and acellular entities from an object or habitat

What are the four ways of sterilization

* Autoclaving
* Dry-heat sterilization
* UV radiation
* Filtration

Describe autoclaving

Steam at 121ºC at 15 psi for 15 minutes, preferred method

Describe dry-heat sterilization

Items in electric oven at 160-170ºC for 2 hours

Describe UV radiation

Can sterilize things, unable to penetrate things.

* Wavenlengths of 260 nm used

Describe filtration

Good for media that can’t be heated, uses bacteriological filter that removes things physically

Define media

Nutrient preparation used for culturing micoorganisms

What are the 3 types of media, what do two of these have in common?

* Broth
* Semisolid
* solid
* Both semisolid and solid have solidifying agent

What is the preferred solidifying agent? Why? What else could you use?

* Agar is preferred as bacteria cannot digest it
* Gelatin could be used, but many microbes can digest it

* What do you call media that is hardened at a slant?


* What about in the upright position?
* What about in a petri dish?

* Agar slant
* Agar deep
* agar plate

Give an example of semisolid media?

Thyioglycolate broth

Describe selective media

Inhibits some microbes and encourages others

Describe differential media

Allows many bacteria to grow, but they grow differently like colorwise, size, etc.

Describe enriched media

Has basic nutrients and is enriched with things like blood or serum. Typically used only for fastidious or difficult to grow microbes

Describe general purpose media

Media used to grow most types of bacteria

What is streak plating used for?

A technique used to isolate pure colonies

Give the general procedure of streak plating

* You swab petri in quadrant 1, flame loop.
* Spread bacteria from quadrant 1 to 2, then flame loop.
* Repeat until all four quadrants have been innoculated

What are serial dilutions used for?

To reduce the population to get separate colonies to plate

Give the general procedure of serial dilutions

* You put one milliliter of the culture in nine milliliters of water and repeat until you get what you want

What is the pour plate technique?

Molten agar and liquid cultures are mixed onto a plate to fix all the cells into place

Plates should have a number between what values to be considered countable?

25-250

What is the difference between dilution and dilution factor

* Dilution: how much of your sample is present (negative exponent)
* Dilution factor: how many times you’ve diluted your sample (positive exponent)

Give the equation for CFU/ml

number of colonies on plate/ (dilution times volume pipetted on the plate)

What is CFU?

Colony forming units, that is the number of microorganisms that form colonies

Give the following information about the hydrogen sulfide test:

* purpose


* media
* What it looks like

* Purpose: Indicates presence/absence of H2S production
* Media: peptone iron agar
* Appearance:
* positive: black line
* Negative: Yellowish, no black lines

Give the following information about the casein hydrolysis test:

* purpose


* media
* reagents
* What it looks like

* purpose: determine if bacteria can degrade casein


* media: Skim milk agar
* What it looks like
* Positive: clearing of cloudy agar
* Negative: Cloudy agar remains

Give the following information about the gelatin hydrolysis

* purpose


* media
* What it looks like

* purpose: Determine if bacteria can degrade gelatin with the gelatinase enzyme


* media: nutrient gelatin tubes
* What it looks like
* Positive: Agar liquifies
* Negative: Agar remains solid

Name all the tests in the imvic tests

* Indole
* Methyl Red
* Vogues Proskauer
* Citrate

Give the following information about the Indole test:

* Purpose
* Media
* Reagents
* What it looks like

* Purpose: detects the breakdown of amino acids like indole
* Media: 1-tryptone broth
* Reagents: Kovac’s reagent
* What it looks like
* Positive: red ring at surface
* Negative: no red ring

Give the following information about the Methyl Red test:

* Purpose
* Media
* Reagents
* What it looks like

* Purpose: Determines the production of mixed Acids
* Media: MVRP broth
* Reagents: Methyl red indicator
* What it looks like
* Positive: Turns red
* Negative: No color change

Give the following information about the Vogues-Proskeur test:

* Purpose
* Media
* Reagents
* What it looks like

* Purpose: Detects products other than acids
* Media: MVRP broth
* Reagents: Barrit’s reagent (AKA VP reagents A and B)
* What it looks like
* Positive: Pink
* Negative: No color change

Give the following information about the Citrate test:

* Purpose
* Media
* What it looks like

* Purpose: Determines ability to use citrate as intermediate in krebs cycle
* Media: Simmon’s citrate agar
* What it looks like
* Positive: Blue
* Negative: no color change

Give the following information about the Catalase test:

* Purpose
* Media
* Reagents
* What it looks like

* Purpose: Determines if catalase is present
* Media: none
* Reagent: hydrogen peroxide solution
* What it looks like
* Positive: Bubbles appear on slide
* Negative: no bubbles

Give the following information about the Oxidase test:

* Purpose
* Media
* Reagents
* What it looks like

* Purpose: Determines if cytochrome oxidase is produced
* Media: Whitman filter paper
* Reagents: oxidase test reagents
* What it looks like
* Positive: purple
* Negative: no color change

Give the following information about the acetamide test:

* Purpose
* Media
* What it looks like

* Purpose: Test an organisms ability to utilize acetamide as deamination
* Media: acetamide agar
* What it looks like
* Positive: purple-red
* Negative: No color change

Give the following information about the Nitrate reduction test:

* Purpose
* Media
* Reagents
* What it looks like after step 1? After Step 2

* Purpose: Determine if bacteria can reduce nitrate
* Media: trypticase nitrate broth
* Reagents: Nitrate reagents A and B, Zinc if needed
* What it looks like
* Step 1
* Positive: Pink
* Negative: no color change
* Step 2
* Positive: no color change
* Negative: pink

Give the following information about the Urease test:

* Purpose
* Media
* What it looks like

* Purpose: Determine if bacteria can use urea for nitrogen source
* Media: urea broth
* What it looks like
* Positive: Deep pink
* Negative: no color change

Define strict/obligate aerobes

Can grow only with oxygen

Define strict/obligate anaerobes

Can grow only in the absence of oxygen

Define microaerophiles

Prefer to grow in small concentrations of oxygen

Define faculative anaerobes

Can grow with or without oxygen, prefer oxygen

Define aerotolerant anaerobes

Can’t use oxygen but tolerates it

Describe the 3 methods of anaerobic culturing

* Boiling media: Removes oxygen from media
* Thioglycollate broth: oxygen is removed from the environment
* Anaerobic jar: removes oxygen after the jar is sealed

Describe the following for carbohydrate fermentation

* Purpose
* Media
* Reaction

* Purpose: Determines if organic acids and gasses are produced
* Media: Glucose broth
* Reaction
* Positive: turns yellow and gas bubble appears for positive acid and gas
* Negative: no color change and no gas bubble

Describe the following for starch hydrolysis

* Purpose


* Media
* Reaction

* Purpose: determines if bacteria can utilize starch
* Media: starch agar plates
* Reaction
* Positive: colorless zone around growth
* Negative: no colorless zone

Give the 5 methods of traditional methods used to identify bacteria

* Streak plate
* Bacterial colony morphology
* Biochemical tests
* Staining methods and microscopy
* Dichotomous keys

Describe the enteropluri-test system

* Multitest system with 12 compartments
* Can identify gram-negative, glucose fermenting, oxidase negative enteric bacteria
* Generates a 5 digit number to identify bacteria

Describe the stapherux test

* used to distinguish staphylocci species based on presence of coagulase and protein A
* Positive Test: clumping, staph aureus
* Negative test: no clumping, staph epidermidis

Describe the rapid strep A

* Identifies infectious strains of strep


* Positive test: color change → Strep A antigens present
* Negative test: no color change

Describe the BIOLOG test system

* Can identify over 1400 bacteria species
* 95 chemical tests

What kind of plate do you use for the kirby-bauer technique?

Mueller Hinton, it’s the best at diffusing antibiotics and grows important bacteria

What is the clear area around the antibiotic disk termed? What do you measure it in?

Zone of inhibition, in mm

What media do we use for environmental testing? Why did we do it?

* RODAC plates, they have a raised surface that can be pressed onto surfaces
* To test environmental samples and the effect of various disinfectants on them

Give the basic method and purpose of testing bacterial mutation

* Uses the gradient plate method, with increasing antibiotic concentration
* Uses Two TSA wedges, the bottom one is plain and the top one with antibiotic
* Those on the high concentration are resistant
* Isolate with kirby bauer

Describe the methods and purpose of the membrane filter technique

* Tests for fecal coliforms by filtering a volume of water
* Tests if drinking water is safe

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How do coliforms vs non-coliforms appear on the media we use for membrane filtering for total coliforms?

On endo agar plates, coliforms are red and metallic

non-coliforms are pink and don’t have a metallic look

How do coliforms vs non-coliforms appear on the media we use for membrane filtering for fecal coliforms?

On MFC agar, fecal coliforms are blue, non-fecal coliforms are a cream to grey color

How do coliforms vs non-coliforms appear on the media we use for membrane filtering for fecal strep?

On KF Streptococcus agar, fecal strep appears pink

How do you calculate coliform colonies per 100 ml

coliform colonies counted times 100 divided over ml of water sample

How do you find FC/FS ratio, and what is it?

Number of fecal colonies/number of fecal strep

Used to determine whether the source of pollution is human or animal feces. The higher the number the more likely it is human

What are the standards for safe drinking water?

* 1 total coliform per 100 mL
* 0 fecal coliforms per mL

List places of the human body with normal flora

* Nose
* Mouth
* Throat
* Ear
* eye
* Stomach
* skin
* Small intestine
* Large intestine
* Urethra
* Vagina

Where are sterile zones of the body?

Brain, most bodily fluids (blood, cerebrospinal fluid) bones

What is blood agar used for?

* What does it detect:
* Appearance:

* Acts as a differential, enriched media
* Throat, nose, and skin
* Shows colony morphology and hemolysis pattern

What is EMB used for?

* What does it detect:
* Appearance:

* Selective and differential
* Rectal culture
* fecal coliforms
* Fecal coliforms appear dark purple
* E. Coli appears green
* Slow and weak fermenters are pink
* colorless are not fecal coliforms

What is TSA used for?

* What does it detect:
* Appearance:

* General media
* Skin

What is snyder agar used for?

* What does it detect:
* Appearance:

* Used to detect susceptibility to cavities
* The more lactose fermenting bacteria, the faster the agar turns yellow, the more prone to cavities

What is tomato juice agar used for?

* What does it detect:
* Appearance:

* Enriched media used to isolate normal flora and pathogenic
* Diversity in mouth

What is sabuourad agar used for?

* What does it detect:
* Appearance:

* Selective media used to detect yeast cells
* White colonies
* Diversity in mouth

Describe hemolysis patterns on blood agar plates

Alpha: Zone of green coloration with indistinct margins

Beta: sharp zone of clear hemolysis

Gamma: no hemolysis

Give the basic method for isolating of soil microbes and antibiotic producers

* Crowded plate technique: get large amount of colonies, but can see zone of inhibition produced by antibiotic producing bacteria
* Take 1g of soil in 99ml blank and mix to get dilutions and isolate antibiotic producers
* Can observe antibiotic activity against various bacteria