micro lab final

if it ends in -ase it is a …

enzyme

if it ends in -ose it is a ….

sugar

what are proteins

long polymers composed of amino acid subunits

what do amino acids consist of

carbon bound by amino/nitrogen-containing group (NH2)

hydrogen

carboxyl group (COOH)

Variable R group

amino acids within the protein are connected by what

peptide bonds

how do you break a peptide bond

hydrolysis

enzyme must use water

OH added to carboyxyl

H added back to amino

once peptide bonds are hydrolyzed, what happens

amino acids can be broken down and used as carbon and energy sources

or reused in anabolic reactions

what is deamination

remove amino group of amino acid

amino group converted to ammonia/urea type

pH lowered- acidic conditions

what is decarbonation

removal of carboxyl group form amino acid

pH is raised

name 2 amino acids that contain sulfur in the R-Groups

cysteine and methionine

what is the purpose of the gelatin hydrolysis test

to differentiate between organisms that produce the exoenzyme gelatinase

what does a positive result for gelatin hydrolysis test look like

remains a liquid at room temp

how to prevent a false positive in gelatin hydrolysis test

chilling the tubes prior to recording result

what is the urea broth test for

determining if microorganism produces urease

exoenzyme that hydrolyzes urea

urease

what is a positive result in urea broth test

hot pink color

what species is referred to as a rapid urease positive

proteus species

what is a reagent in the phenylalanine slant

ferric chloride

what is the end product in a phenylalanine slant

phenylpyruvic acid

what is the enzyme in the phenylalanine slant

phenylalanine deaminase

what is a positive result for the phenylalanine slant

orangish yellow to green

what is the ornithine decarboxylase differential for

gram negative enteric bacilli

ornithine decarboxylase tests for what enzyme

ornithine decarboxylase

what is a positive result for ornithine decarboxylase test

yellow to purple/grey

how to do/read a ornithine decarboxylase test

transfer 200ul of bacteria into broth

overlay tube with mineral oil

incubate

observe lower half for color change

growth must be present for analysis

What does SIM agar deep test for

sulfur, indole, motility

what is IMViC

indole production, methyl red, Voges-Proskauer, citrate utilization

used to differentiate enteric bacteria

what does sulfur reduction look like in SIM deep

black precipitate

in the SIM deep indole production is a result of the breakdown of what amino acid

tryptophan

what reagent must be added to SIM deep indole test

Kovac’s reagent

what is a positive test in SIM deep indole production

cherry red ring

In the SIM deep what is a positive result for motility

growth outward from the initial stab line

Oxidation Vs Reduction

O- removal of electrons

R- Gain of electrons

what is ATP

energy source for all living organisms

what is aerobic respiration

Metabolism occurs in the presence of oxygen

what is anaerobic respiration

metabolism occurs in the absence of oxygen

what is facultative respiration

metabolism occurs in both the presence and absence of oxygen

what are the terminal/final electron acceptors for aerobic and anaerobic respiration

aerobic- Oxygen- yields most ATP

anerobic- CO2, sulfate, nitrate

oxidative vs fermentative

O- oxygen required

F- oxygen not required

Toxic byproduct examples

superoxide

hydrogen peroxide

hydroxyl

what are reactive oxygen Intermediates (ROI)

Damage DNA and results in accumulation of mutations that are detrimental

what are obligate aerobes

bacteria that require oxygen for growth

what are obligations anaerobes

bacteria that will only grow in the absence of oxygen

what are microaerophiles

organisms that require a limited amount of oxygen to grow

7-10% oxygen

what are aerotolerant anaerobes

cannot use oxygen for growth but are not killed by it

produce catalase and can detoxify ROI’s

what are facultative anaerobes

grow best with oxygen but can live without it

what does a positive thioglycolate broth result for aerobes look like

only growth in the top portion of tube

what does a positive thioglycolate broth result for obligate anaerobes

growth only at the bottom of the tube

what does a positive thioglycolate broth result for microaerophiles

seen towards the top of the tube but below the surface

what does a positive thioglycolate broth result for facultative anaerobes

growth throughout tube and cloudy at top

what does a positive thioglycolate broth result for aerotolerant anaerobes

middle of the tube to bottom mainly, cloudy near bottom

explain how a GasPak works

in a sealed plastic bag you have your plate, a catalyst pack, and a wet paper

the pack contains a palladium catalyst that in the presence of water will produce CO2&H

H will combine with water to form more water until there is no more oxygen present

This creates an anaerobic environment

what is the oxidative test for

to see if the organism is capable of aerobic respiration/ if cytochrome oxidase is present

what is the reagent in oxidase test

chromogenic reducing agent

what is a positive result of oxidase test

dark blue when oxidized

when can a false positive result in the oxidase test occur

reading test after 30 seconds

using a non clear oxidase reagent

what does the catalase test fo

enzyme catalase

what is a positive result in catalase test

bubbles

reagent in catalase test

hydrogen peroxide

what does a nitrate broth test for

if an organism can reduce nitrate (NO3) to nitrite (NO2) to ammonia (NH3)

what are the reagents in the nitrate reduction test

Nitrate reagent A

Nitrate reagent B

Zinc dust

what is the enzyme in nitrate reduction test

nitrate reductase

Inoculation protocol for nitrate reduction test

using P200 pipet transfer 200uL of organism into nitrate broth

protocol for adding reagents to nitrate broth

after incubation add 5 drops of nitrate reagent A and 5 of B

finger vortex

wait two min and if no color yet add a very small dusting of zinc using tweezers

positive result for nitrate reduction test

red color- 2 reagents

not red- zinc dust

do feces cause disease

no indicator organisms do

what are coliforms

indicator organisms most frequently used in water quality assessment

characteristics of coliforms

aerobic or facultative anaerobes

gram -, rod-shaped bacteria

ferment lactose

not usually pathogens

indicates fecal contamination

what are the 3 stages of coliform testing

presumptive test

confirmed test

determination of # of coliforms

what is the presumptive test for coliforms

most probable number method

presumptive test positive result

red to yellow color change in the presence of bubbles

confirmation test uses what agar

MUG agar

what is the MUG agar differential for

GUD enzyme

what is a positive result for confirmation test

fluorescent compound visible in UV light

what other media can be used for a confirmation test

EMB agar

what is most probable number

the ratio of the number of lactose fermentative tubes giving a positive result

what is multiple tube technique

series of dilutions

3 test tubes - 10ml, 1ml, .1ml

10ml tube has 2x strength

EPA recommendations for standard drinking water

less than 1 colony per 100ml

EPA recommendations for domestic water supply

2000 colonies per 100ml

EPA recommendations for fishing/boating

1000 colonies per 100ml

EPA recommendations for body contact recreation

100 colonies per 100ml

sources of food contamination

improper handling and preparation

or food-processing and distribution errors

common sources of infection

Bacillus cereus,

Shigella,

Cyclospora (protist),

Cryptosporidium (protist)

3 basic media types

selective- prevents growth of unwanted bacteria

enriched- for fastidious organisms

differential- contains factors some bacteria can change in a recognizable way

techniques to test for food contamination

evaluated for coliforms

standard plate count- serial dilution + spread plating

why is 5-second rule a myth

bacteria transfer to food instantaneously upon contact, regardless of time

what is SS agar selective/differential for

enteric pathogenic bacilli

S- Bile salts and dyes, inhibit gram + and most gram-

D- Lactose (neutral red), Sodium thiosulfate and Ferric citrate (black centers)

colony morphology and results for ss agar plates

colorless or red colonies with possible black centers

what is EMB agar selective and differential for

S- eosin and methylene blue dyes inhibit gram +

D- Lactose fermentation (pink +) (colorless -), strong acid production - greenish sheen

what is MSA selective and differential for

S- Gram + (staphylococcus spp) halophiles

D- presence of mannitol (S. aurous), acid production (yellow)

sterile

Completely void of any viable organism; both inside and outside

Bacteriostatic

Methods used simply to inhibit and minimize growth amounts

Bactericidal

Methods to completely eradicate viable cells

what is important to consider in heat sensitivity

type of object/product

mechanism for heat sensitivity

– coagulation of proteins

– accumulation of free radicals (DNA damage)

what is heat sensitivity used for

Sterilization of glassware, etc. (things that tolerate high heat without damage)

drawback of heat sensitivity

• slower penetration or heat transfer

• not suitable for all materials - some will melt or degrade

Certain plastics will degrade and crumble after so many rounds of high heat

Psychrophilic

0°C – 15°C

Mesophilic:

20°C – 40°C

Thermophilic:

45°C – 65°C