Gen Microbio LAB

What’s the significance of the parfocal microscope?

it keeps the slide in focus when you switch between objective lens

How should you start using a microscope?

by always starting on the lowest power

How do you use the oil immersion?

• wait until image is focused and centered from the previous lens objectives

• never drop back down to a lower magnification

What should you never discard in lab?

any items made of glass

How to handle live cultures?

always wear gloves and use proper aseptic technique

What’s the primary method to fix bacteria?

heat fixing

Broth cultures

media that provides large numbers of bacteria in a small space and are easily transported

Agar slants

test tubes containing solid culture media that were left at an angle while the agar solidified; they provide a solid growth surface and are easier to store/transport

Agar deep

test tubes with agar that was allowed to solidify in the bottom; used to grow bacteria that require less oxygen

What pattern of growth is on this agar slant?

Arborescent (branched)

What pattern of growth is on this agar slant?

Beaded

What pattern of growth is on this agar slant?

Echinulate (pointed)

What pattern of growth is on this agar slant?

Filiform (even)

What pattern of growth is on this agar slant?

Rhizoid (rootlike)

What pattern of growth is on this agar slant?

Spreading

Why do we fix bacteria?

in order to kill the bacteria and prevent it from causing cells to break and it also preserves microbes

What are 2 methods for fixing bacteria?

heat-fixed OR use of methanol/alcohol

Why do we stain bacteria?

so it can be clearly seen and identified udner a microscope

Simple Stains

staining procedure that uses only 1 stain; used to determine cell mmorpholgy, size & arrangement

Basic Stain

when the chromosphere is a positive ion

Acidic Stain

when the chromosphere is a negative ion

Direct Stain

a simple stain that stains the bacteria itself

Negative Stains

a simple stain that stains the background but leaves the bacteria; used for difficult-to-stain coccobacilli

What BioSafety Level (BSL) is our lab?

1

Gram Stain

a differential stain that enables you to classify bacteria as either pos or neg

What are the steps in Gram staining?

1. apply primary stain

2. apply mordant

3. apply decolorizing agent

4. apply secondary OR counterstain

Primary Stain

1st step of gram-staining; crystal violent used to stain all bacteria purple

Mordant

2nd step of gram-staining; Gram’s iodine used to combine with crystal violet in cell wall to form CV-I

Decolorizing Agent

3rd step of gram-staining; ethanol/ethanol-acetone used to wash out out the primary stain of some bacteria while others are unaffected

Secondary Stain (“counterstain”)

4th step of gram-staining; safranin is used to stain the decolorized bacteria red

Gram (+) bacteria

contain a thick peptidoglycan cell wall that better traps the dye color; cell looks purple/blue

Gram (-) bacteria

have a thin peptidoglycan cell wall that can’t trap the dye color; cell looks pink/red

Streak Plate Technique

inoculating loop is used to spread a sample many times over a solid medium; causes bacteria to fall off loop

How does nutrient gelatin work?

it solidifies when cooled below 25C and liquefies when heated to 25C

Litmus milk

skim milk w/ the pH indicated litmus added

Urea

a waste product of protein digestion in most invertebrates; usually excreted in urine

Urease

splits ammonia from urea

Urea agar

used to detect bacteria that can produce urease

Litmus test turns PINK when what?

if the bacteria can ferment lactose

Litmus test turns PURPLE when what?

if the bacteria can break down amino acids

Litmus test turns WHITE when what?

the bacteria can reduce litmus

What shows a urea test is negative?

yellow color

What shows a urea test is positive?

pink color

What shows a gelatin hydrolysis test is positive?

bacteria causes it to liquefy

What shows a gelatin hydrolysis test is positive?

bacteria causes it solidify

Deamination

the removal of an amine group from a molecule

Decarboxylation

the removal of a carboxyl group (CO2) from an AA

Decarboxylases

enzymes that break down decarboxylations

MIO Medium (Deep)

Motility, Indole production, and Ornithine decarboxylase activity

• tests for ability of bacteria to deaminate tryptophan

Selective Media

medium that contains chemicals that prevent the growth of undesirable media w/o interfering w/ growth of desired media

Differential media

medium that contains compounds that result in visible differences btwn bacterial species

Starch

a polymeric carbohydrate consisting of a large # of glucose units joined by glycosidic bonds

Amylases

enzymes that break down starches into sugars

Exoenzyme amylases

produced by bacteria; breaks down large molecules into smaller units that can be inserted into bacteria

Starch Agar

type of differential media; a nutrient agar enriched w/ starches

Oxidative Catabolism

requires O2; CO2 and H20 are common byproducts

Fermentative Catabolism

doesn’t require O2 BUT can occur in its presence; byproducts are usually small organic molecules

What shows a positive in Starch Hydrolysis test?

presence of zone formation

What shows a positive in OF-Glucose test?

yellow color in both aerobic & anaerobic chamber

What shows a negative in OF-Glucose test?

green or blue color in anaerobic chamber; blue color in aerobic chamber

What’s the purpose of the “pinch” in the tube containing the OF-glucose media?

it creates the aerobic & anaerobic conditions

Acetoin

neutral product made by fermentative bacteria during sugar metabolism

Citrate Test

detects the ability of an organism to use citrate as the sole source of carbon & energy

What shows a positive in Methyl Red test?

red solid color on top

What shows a positive in Voges-Proskauer test?

red color or band at the top; shows acetoin is present

What shows a positive in Citrate test?

blue color on top of slant

Nitrate Reduction Test

used to determine ability of bacteria to reduce nitrate

What’s a negative result for both aspects of a Nitrate Reduction Test?

pale orange/peach color in broth

What’s a positive result of a Nitrate Reduction Test?

deep red color in broth

Oxidase Test

used to detect presence of cytochrome c in bacterium

Positive result of Oxidase Test?

blue color visible in petri dish; also means that bacteria is aerobic

Catalase Test

1 of 3 main tests used to identify bacteria speciies

Positive result of a Catalase Test?

visible rapid bubble formation; means bacteria is STAPHYLococci

Negative Result of a Catalse Test?

no change occurred; means the bacteria is STREPTOcocci

Which UV range is most damaging

100-280nm

Why are free radicals dangerous?

they have unpaired electrons, making them very unstable

Light Repair

photolyses SPLIT the dimers

Dark Repair

dimers are removed by endoculeases

Catalase

enzyme that breaks down H2O apart

Nitrate Broth

used to determine if a bacteria species can reduce nitrates

Ionizing Radiation

carries enough energy to knock liberate electrons from atoms/molecules

• ex: gamma rays, x-rays, extreme UV

Non-Ionizing Radiation

doesn’t carry enough energy to ionize an atom/molecule BUT does have enough energy to cause excitation

• ex: UV, visible light, IR, microwave, radio waves