question

the eyepiece is the _______ lens

answer

ocular (10x)

question

the lenses on microscope (4 x, 10 x, 40 x) are called ____________ lens

answer

objective

question

_________ is for mounting specimen

answer

stage

question

_________ oil is used to minimize __________

answer

immersion oil, light scattering

question

remove scope from cabinet using_____ and ______

answer

arm and base

question

use ______ adjustment knob first but do not hit the ______ lens

answer

course
objective

question

only use _______ adjustment once you have found object

answer

fine

question

what objective lens do you use oil for

answer

100 x

question

you use ________'s to wipe oil off the lens

answer

kimwipe

question

rod shaped-
shepre shaped-
yeast

answer

rod shaped-bacilli
sphere-cocci
yeast- unicellular circles/ovals

question

the method of handling microbes and materials in a way that minimizes contamination is called _______ technique

answer

asceptic technique

question

the process of transferring a microbe from one medium to the next is called _______ and the sample being transferred is called an __________

answer

inoculation
inoculum

question

forms of media

answer

1. liquid: nutrient broth in tube (NB)
2. solid: nutrient broth+ agar (solidifying agent)(NA-nutrient agar)

question

deep, slant, plate/petri dish

answer

deep- liquid straight across
slant- semi solid at a slant
petri dish- plate

question

transferring an inoculum using aseptic technique

answer

-inoculate 2 NA slants from 2 NB cultures (take culture from 2 nutrient broths and inoculate them into slants)
-inoculate 1 TSB from 1 NA plate (take culture from plate and inoculate into TSB (soy broth)
-inoculate 1 NA plate from 2 broth cultures (divide plate) (take cultures from 2 broths and inoculate plate, one culture per side)

question

the loop we flame is

answer

an inoculating loop

question

when do you need to sterilize

answer

-every time you open a test tube, the mouth must be sterilized
- before replacing cap, flame again
-flame loop before and after use

question

the _____ is always used when transferring organisms from a liquid medium

answer

loop

question

streak plate technique

answer

- isolation streak plate is a method to separate individual bacteria from mixtures
-when a mixture of bacteria is streaked across a plate, the concentration of bacteria gets so low that a single bacterium can form isolated colonies on the surface of the plate
- a colony arises from a single bacterium that divides repeatedly to form a visible mass of cells

question

isolation streak plate is a method used to separate individual_________ from _________

answer

bacteria from mixtures

question

streak plate steps

answer

1. flame loop, dip in inoculum, use to streak section A
2. flame loop between A and B, use streaks in section A as the inoculum for section B
3. it is not necessary to flame loop between section B and C, use streaks in section B as inoculum for section C a ^b>cv

question

following incubation of streak plate you should observe

answer

the presence of individual colonies

question

simple staining

answer

- useful for observing shape and arrangement of cells
-use only one stain

question

if only the cells are stained then it is called a ________ stain, if the background is colored, leaving some or all of the cell structure unstained, then it is called _______ stain or also known as ______ stain

answer

just cells= DIRECT stain
background colored leaving some or all of cell structure unstained= NEGATIVE STAIN

question

difference between direct and negative stain

answer

-direct stain directly stains cells
-negative stain colors background and leaves part or all of cell unstained

question

capsule stain is an example of

answer

negative stain (makes capsule show up, rest of slide colored)

question

capsule is also known as
what functions?

answer

glycocalyx
-makes cell sticky helping them attach better to surfaces
-they evade host immune system

ex: streptococcus pneumoniae, mycobacterium

question

smear preparation and staining for DIRECT SIMPLE STAIN

answer

-get clean slide
-add lapful of water to slide
-obtain TINY amount of bacteria from plate using loop
- mix bacteria into drop of water and spread mixture
-let the mixture air-dry completely
-pass the slide through the flame 3 times to HEAT FIX
-cover smear with CRYSTAL VIOLET for 1 min
-rinse smear until run off it clear and blot dry
-observe using all lenses

question

why do we heat fix when doing a smear and stain

answer

1: causes the cells to adhere to the slide so it wont wash off
2:Kills the microorganisms
3:Causes changes in the bacterial cells which causes them to stain better

It removes water from the bacterial cell, pores open in the cell so more stain can enter the cell wall.

question

if bacterial cells aren't killed before smear what might happen

answer

their ability to retain dye will not be as good as it would if they were dead

question

what is the goal of streaking a plate?

answer

to produce isolated individual colonies on the agar plate

question

what is a bad streak plate?

answer

one that shows bad flaming after area A (too much bacteria)
improper set up

question

how to inoculate a streak plate? explain and draw

answer

1. open tube of nutrient broth/organism
2. flame tube mouth
3.flame loop
4. obtain some of the organism
5.make streak in section A
6. flame loop
7. use section A as medium for section B
8. dont flame
9.use section B for section C

question

what would you expect to see if you forgot to heat fix your slide?

answer

nothing, bacteria would wash off the slide if it isn't heat fixed!!
if it didn't wash off it wouldn't retain dye very well either.

question

differential staining (3 types)

answer

useful for the identification of specific groups of organisms
* uses two or more stains to complete the procedue
1. gram stain
2. endospore stain
3. acid-fast stain

question

gram stain

answer

-based on the cell wall characteristics that differentiates two major groups of bacteria
-gram +(purple) and gram - (pink)

question

how to do gram stain

answer

1.make smear
2. cover smear with crystal violet for 1 min, rinse with water
3. cover smear with iodine for 1 min, rinse with water
4. tip the slide and drip alcohol down until run off is clear (10 seconds!)
5.cover smear with safranin for 1 min
6. rinse with water and blot dry

CRYSTAL VIOLET(primary), IODINE(mordant), ALCOHOL(decolorization),SAFRANIN (counterstain/pink)

question

what order do we use the dyes and alcohols for gram stain

answer

crystal violet
iodine
alcohol
safranin

question

the primary stain in gram staining isthe mordant isthe decolorization isthe counterstain is?

answer

primary-crystal violet
iodine- mordant
alcohol- decolorization
safanin- counterstain

question

differential staining (3 types)

answer

useful for the identification of specific groups of organisms
* uses two or more stains to complete the procedue
1. gram stain
2. endospore stain
3. acid-fast stain

question

gram stain

answer

-based on the cell wall characteristics that differentiates two major groups of bacteria
-gram +(purple) and gram - (pink)

question

how to do gram stain

answer

1.make smear
2. cover smear with crystal violet for 1 min, rinse with water
3. cover smear with iodine for 1 min, rinse with water
4. tip the slide and drip alcohol down until run off is clear (10 seconds!)
5.cover smear with safranin for 1 min
6. rinse with water and blot dry

CRYSTAL VIOLET(primary), IODINE(mordant), ALCOHOL(decolorization),SAFRANIN (counterstain/pink)

question

what order do we use the dyes and alcohols for gram stain

answer

crystal violet
iodine
alcohol
safranin

question

the primary stain in gram staining isthe mordant isthe decolorization isthe counterstain is?

answer

primary-crystal violet
iodine- mordant
alcohol- decolorization
safanin- counterstain

question

endospore

answer

clostridium and bacillus produce endospores
-endospores- a survival structure that allows the bacterial cell to survive unfavorable conditions
- under favorable conditions they germinate and release a viable vegetative cell

question

acid-fast stain

answer

-can differentiate between acid fast- and non acid-fast bacteria
-acid fast have mycolic acid in cell walls making them waxy

question

what genus can be recognized using the acid fast stain?

answer

mycobacterium (have my colic acid)

question

fungi
examples

answer

eukaryotic
unicellular (yeast)
or
multicellular (molds)

mold ex: asperfillus, rhizopus, penicillium

question

protozons

answer

eukaryotic
-divided into 4 groups based on motility:
1. use cilia (paramecium)
2.use amoeboid motion (amoeba)
3. use flagella
4. no motility

*know how paramecium and amoeba look

question

fungal infections like athlete's foot are challenging to treat and often require long term courses of anti fungal drugs. why do you think fungal infections are so difficult to treat?

answer

because both fungal and human cells are eukaryotic so this makes them have less differences that can be targeted without damaging our own cells.

question

kinds of media

answer

selective media- supports growth of one group of organisms but inhibits the growth of another. usually they contain salts or dyes to inhibit the growth of the organisms not targeted for study

differential media- allows the growth of more than one microorganism of interest but with distinct differences in appearance of colonies. usually contain a specific substrate and pH indicator to serve as a differential medium.

selective and differential media- allows for only certain bacteria to grow, with a component that also differentiates among the species that survive.

enriched media- extra nutrient constituents have been provided to support the growth of fastidious microbes or encourage the growth of particular bacteria

question

what media allow certain bacteria to grow, but also has component that differentiates among those bacteria that have survived

answer

selective & differential media

question

what media supports the growth of one group of organisms while inhibiting the growth of another (usually contain salts or dyes to inhibit the growth of the organisms not targeted)

answer

selective media

question

which media tends to use salts or dyes to inhibit growth of organisms

answer

selective media

question

which media allows for the growth of more than one microorganism of interest but with distinct differences in appearance of colonies (usually uses a specific substrate and pH indicator to serve as a differential medium)

answer

differential media

question

in differential media what is usually used as a means of differentiation

answer

pH indicator or specific substrate

question

what kind of media provides nutrients to support the growth of fastidious microbes or encourage the growth of particular bacteria

answer

enriched mediaa

question

blood agar

answer

-both enriched and differential medium
-we can differentiate kinds of streptococci based on their patterns of hemolysis
-hemolysins- toxins that can break down red blood cells
-kinds of hemolysis:
1. beta hemolysis: complete breakdown of the RBC's (clearing around colonies)
2. alpha hemolysis: partial destruction of the RBCs (greenish brown color around the colonies)
3. gamma (y) hemolysis: no lysis of RBS (intact blood agar around colonies)

question

blood agar is what kind of medium

answer

enriched and differential

question

hemolysins

answer

toxins that can break down red blood cells

question

how can we differentiate different kinds of streptococci?

answer

on their patterns of hemolysis

question

what are the patterns of hemolysis

answer

beta (?) hemolysis: complete breakdown of the RBCs (clearing around the colonies)
alpha (?) hemolysis: partial destruction of the RBCs (greenish brown color around the colonies)
gamma (?) hemolysis: no lysis of RBC (intact blood agar around the colonies)

question

which hemolysis causes clearing around colonies

answer

beta hemolysis (complete breakdown of RBCS)

question

which hemolysis causes greenish brown color around colonies

answer

alpha hemolysis- partial destruction of RBC

question

which hemolysis leave blood agar around colonies intact?

answer

gamma (y) hemolysis- no lysis of RBC

question

mannitol salt agar medium

answer

-selective and differential media
-shows staphylococcus pathogenic and nonpathogenic
-- selective: high concentration of salt selects salt tolerant (halophilic) satphylococcus only
-differential: mannitol fermentation differentiates pathogenic and nonpathogenic staphylococcus
- pathogenic species of staphylococcus: grow and ferment mannitol to acid- phenol red (pH indicator) changes from pink to yellow
-non pathogenic staphylococcus: grow but can't ferment mennitol-indicator stays pink

question

mannitol salt sugar has what medium

answer

- selective and differential
- selective: high concentration of salt selects salt tolerant (halophilic) satphylococcus only
-differential: mannitol fermentation differentiates pathogenic and nonpathogenic staphylococcus

question

how do we

answer

question

staphylococcus is ___________ or has a high concentration of salt so it is selected this way against other bacteria using the mannitol salt agar (selective media)

pathogenic species of staphylococcus grow and ______ mannitol to acid which causes indicator to turn from pink to ________

nonpathogenic species of satphylococcus: grow but can't _______mannitol- indicator ________

answer

halophilic

pathogenic-ferment manitol, change pink to yellow

nonpathogenic- grow but can't ferment manitol, indicator stays pink

question

eosin methylene blue agar

answer

-selective and differential medium
-selective: dyes 1. eosin and 2.methylene blue select for gram negative bateria (including coliforms)
-differential: lactose fermentation differentiates between coliforms (lector fermentors) and noncoliforms (non lactose fermenters)

coliforms- grow and ferment lactose to acid- trigger a pink to metallic green colony coloration
-non-lactose fermenters: colonies will appear colorless

SOOO
gram positive: no growth
lactose fermenter: pink
non lactose fermenter: colorless
ecoli: metallic green (coliform)

question

what differentiates between coliforms and noncoliforms?

answer

-coliforms=lactose fermentation

-non coliforms=non lactose fermenters

question

coliforms

answer

coliforms- grow and ferment lactose to acid- trigger a pink to metallic green colony coloration
-non-lactose fermenters: colonies will appear colorless

question

coliforms which grow and ferment lactose to acid will trigger a _____ to ____ colony coloration on eosin methylene blue agar

answer

pink to metallic green

question

non lactose fermenters will appear _____ on eosin methylene blue agar

answer

colorless

question

macconkey agar

answer

-nosocomial infections: infections acquired in a hospital setting ex: ventilator associated pneumonia (VAP)
-common causes of VAP: gram positive organisms such as streptococcus, staphylococcus, gram negative organisms including coliforms ( such as ecoli) as well as non coliforms (pseudomonas)
-media to distinguish the above scenario- a good tool to have clinically
-maconket agar: SELECTIVE AND DIFFERENTIAL MEDIUM

-selective: gram positive
-differential: coliforms, non coliforms.

selective: bile salts and crystal violet select for gram negative bacteria (including coliforms)

differential: lactose fermentation differentials between coliforms and noncoliforms (lactose fermenters- pink, non lactose fermenters: colorless)

question

ecoli is gram _____ and is a _____

answer

gram negative, coliform

question

what selects for gram negative bacteria?

answer

bile salts and crystal violet

question

macconkey agar is for

answer

-identifying gram negative coliforms and noncoliforms to identify causes of nosocomial infections/ventilator associated pneumonia

question

standard plate count

answer

-commonly used method to estimate the number of microbes in a sample
-originial smaple is diluted through a series of bottles/tubes
-a volume of the dilution is put onto plates and number of colonies are counted after incubation
-number of bacteria in the original sample is calculated by multiplying number of colonies on the plate by the dilution factor corresponding to that plate
-only plates containing between 30 and 300 colonies are considered to be valid and countable

question

only plates containing between _ and _ colonies are considered valid and countable

answer

30 and 300

question

The presence of coliforms in food or water indicates the possibility of fecal contamination and thus the potential of fecal pathogens also being present.

answer

true

question

Both MacConkey agar and EMB are useful in growing Gram positives.
true or false

answer

false- both select for gram negative

question

high concentration of ______ in mantol salt agar makes this medium selective, while the ________ makes it differential. fermentation of mantel results in the medium around the colony changing from ____ to _____

answer

salt makes it selective
mantel makes it differential
red to yellows

question

growth of ecoli on EMB (eosin methylene blue agar) would produce _________ colonies while growth on the maconkey agar would produce ______ colonies

answer

green
pink

question

ou are testing a sample of water from a local swimming pool. Your 10-1 final dilution plate has 38 colonies on it. The safety standards from swimming pools state that the water cannot contain more than 200 CFU per milliliter [cells per milliliter]. Is the pool you are testing safe to swim in? Why or why not?

answer

b. No, the final cell number is 380 and therefore, more than 200 CFU per milliliter. Correct

question

You are testing a sample of milk from a New York dairy that has been cited for some recent safety violations. Your 10-2 final dilution plate has 201 colonies on it. The FDA safety standard for milk of the type you sampled is 3x105 per milliliter. Is this milk sample safe to drink? Why or why not?

answer

b. Yes, the final cell number is 2.01x104 and, therefore, less than 3x105 per milliliter. Correct

question

blood agar results

answer

patient A- complete clearing beta hemolysis
streptococcus pyrofens: complete clearing
pseudomonas aeruginosa: incomplete clearing alpha hemolysis
staphylococcos epidermidis: no clearning gamma hemolysis

question

macconkey results

answer

sample I- colorless colonies (non lactose)
e coli- pink colonies (lactose fermenter)
pseudomonas aeruginosa: colorless colonies
staphylococcus epidermidis: no growth

question

manitol salt agar results

answer

sample m- color around colony yellow (mannitol fermenter) pathogenic sp

staphylococcus aureus: color around colony yellow (mannitol fermenter) pathogenic sp

staphylococcus epidermidis- growth but no color change (non mantel fermenter) non pathogenic sp

ecoli- no growth

question

eosin methylene blue results

answer

sample p: metallic green colonies (lactose fermenter)
ecoli- metallic green colonies (lactose fermenter)
pseudomonas aeruginosa: colorless colonies (nonlactose)
staphylococcus epidermidis: no growth

question

aerotolerance

answer

oxygen requirement of bacteria varies dramatically
a. obligate aerobe- growth at top of tube only where high oxygen concentrations have diffused into medium

b. facultative anaerobe- both aerobic and anaerobic growth but greater growth with oxygen, growth is best where the most oxygen is present at top but occurs throughout tube

c. obligate anaerobe- only anaerobic growth, ceases in presence of oxygen, growth occurs at bottom where there is no oxygen

d. aerotolerant anaerob- only anaerobic growth, but continues in presence of oxygen, growth occurs evenly and oxygen has no effect

question

for what microbes does growth occur evenly, and oxygen has no effect

answer

aerotolerant anaerobes

question

for what microbes does growth occur most at bottom of tube

answer

obligate anaerobes- they cannot grow where there is oxygen

question

for what microbe does growth occur most at top of tube but still is present through out

answer

facultative anaeobe- does but aerobic and anaerobic growth but greater growth in presence of oxygen

question

for what microbe is growth only occurring at top of tube where oxygen is

answer

obligate aerobe

question

grwoth patterns in BHI adar deeps

answer

brain-heart infusion nutrient agar

tube 1: obligate aerobe: growth is only at the surface o the tube
tube 2: facultative anaerobe: uneven distribution of growth from top to bottom (more growth at top)

tube 3: aerotolerant anaerobe: uniform growth from top to bottom

tube 4: obligate anaerobe: absence of growth in the top portion of tube where oxygen is present. also not large cracks in the agar as a result of gas production by the anaerobic organism

question

cracks are shown in the agar as a result of gas production by the ______ micro organism

answer

obligate anaerobic

question

catalase

answer

-catalyzes hydrogen peroxide to water and oxygen
-hydrogen peroxide is toxic to the cells so many bacteria produce the enzyme catalase to break it down
- this test helps in distinguishing gram positive cocci from one another:
staphylococcus- catalase positive, white film
enterococcus- catalase negative, more clear

question

oxidase (cytochrom c oxidase)

answer

-cytochrome c oxidase is the final enzyme of the electron transport chain that reduces O2 to form H2O
-purpose of this test is to determine if the bacterium of interest has cytochrom C oxidase in its electron transport chain
-this helps distinguish gram negative rods from one another

question

what helps us distinguish gram positive cocci from one another?

answer

catalase test

question

what helps us distinguish farm negative rods from one another?

answer

cytochrome c oxidase test
(aerobic organisms like pseudomonas test oxidase positve) (facultative anaerobes such as E.coli- test oxidase negative)

question

what acts as final electron acceptor in anaerobic respiration

answer

nitrate, sulfate, sulfur, and carbonate

question

what acts as the final electron acceptor in fermentation

answer

pyruvate and pyruvate derivatives

question

coal tar

answer

coal tar derivatives have been shown to cause cancer in animals but are allowed in cosmetics because they are only applied to the skin and aren't being injected into humans. coal tar colorants are listed as:

FD&C- color that can be used in food, drugs, and cosmetics
D&C- color that can be used only in drugs and cosmetics
External D&C- color that can be used in drugs and cosmetics applied to skin surface

question

why is proper storage a good idea

answer

1. increased heat causes breakdown of cosmetic
2. degraded ingredients may irritate skin.

question

non comedogenic

answer

will not close the pores of the skill, so it should be less likely to cause acne

question

nutrient agar (NA)

answer

-general purpose culture medium
-rich in nutrients

question

mannitol salt agar (MSA)

answer

-red
-high salt- inhibits most bacteria (NOT staphylococci)
-mannitol- sugar alcohol- bacteria which can ferment this produce acid that causes a yellow color change in media.
-NON fermenting- red, FERMENTING- YELLOW
-staphylococcus aureus- causes infections such as acne to abscesses, toxic shock, blood poisoning.

question

what is the name of the bacteria causing acnes, abscessed, toxic shock, blood poisoning which we use the __________ to test?

answer

MSA-- mannitol salt sugar

staphylococcus aureus

question

staphylococci that ferment mannitol will produce a _________ color change

answer

YELLOW (MSA PLATE)

question

non- mannose fermenting bacteria produce what color in the MSA plate

answer

red

(yellow if it DOES ferment mannitol)

question

eosin methylene blue agar (EMB)

answer

-contains a purple dye INHIBIITING gram positive -SUPPORTS GRAM NEGATIVE GROWTH -lactose fermenting bacteria metabolize the lactose in the media and produce acid causing a color change (GREEN for strong acid such as ecoli or PINK for weaker fermentation)

question

to test for presence of fungi in producets use

answer

sabouraud dextrose agar

question

sabouraud dextrose agar ((SDA)

answer

- a medium used for growing fungi because the nutrients and slightly acidic environment encourages fungal growth and survival
- fungi such as yeast and molds grow

question

what does MSA select for
what does it differentiate

answer

staphylococci
manitol and non manitol fermenters

YELLOW if fermented

(manitol fermenters are pathogenic) staphylococcii aureus

question

what does EMB select and differentiate for

answer

selects for gram -
differentiates lactose fermenters and nonlactose fermenters/coliform/noncoliform

GREEN if coliform
coliform- ecoli feca matter

question

macconkey selects and differentiates for

answer

selects- gram -
differentiates:: lactose fermenters and non lactose fermenters
coliform/noncoliform

* test nosocomial infections due to hospital like ventilator associated pneumonia

RED if coliform

question

what tests nosocomial infections like VAP

answer

maccconkey

question

which tube would have cracks seen due to gas production

answer

facultative anaerobe
aerotolerant anaerobe

question

catalase distinguishes what

answer

gram positive cocci

h202> h20 and oxygen

question

oxidase disttinguishes what

answer

gram negative rods

question

does staphylococcus contain catalase

answer

yes (film shown)

question

does enterococcus contain catalase

answer

no (clear)

question

what is the final enzyme of ETC

answer

oxidase (reduces O2 to H2O)

question

will aerobic have oxidase

answer

YES
anaerobic WILL NOT

question

what is blood agar....selective or diff

answer

differential !!

question

pink cells with green stained structure

answer

endospore stain

question

what is the purpose of performing an isolation streak plate

answer

to obtain a pure culture or to determine if a culture is pure

question

two characteristics of a hypothesis

answer

must be falsifiable, makes a prediction between 2 variables

question

Students commonly confuse Saccharomyces cerevisiae and Staphylococcus Aureus when viewed on a microscope slide. How could you microscopically differentiate S. Cerevisiae and S. Aureus?

answer

S. cerevisiae will be LARGER

question

Considering the Gram stain technique, what would you observe if you decolorized too much?

answer

all cells decolorized

question

final dilution

answer

multiple dilution factors for all tubes (by adding exponents)

question

oxidase presence causes what

answer

purple color change

question

ecoli is a _________ ____________

answer

facultative anaerobe

question

kirby-bauer method

answer

used to test sensitivity of bacteria to antibiotics
larger zone of inhibition= more effective
- measure in mm

question

what are the biochemical tests

answer

-gelatin hydrolysis
- starch hydrolysis
- casein hydrolysis
- carbohydrate formation (phenol red)
- TSI
- IMViC reactions (SIM, MRVP, Simmons citrate)
- urease test
- nitrate reduction

question

gelatin hydrolysis

answer

- protein GELATIN is broken down by enzyme GELITINASE
- gelatin medium is semi- solid at room temp and solid when refrigerated
- inoculated with organisms, incubated at 37C, and then refrigerated

POSITIVE FOR GELITANASE- tube is runny after incubation/ refrigeration

NEGATIVE FOR GELITINASE- tube gels after inoculation and refrigeration

question

if you inoculate a gelatin medium and refrigerate it and it comes out runny what does this mean?

answer

the organism is gelatinase positive because the enzyme has broken down the gelatin

question

if you inoculate a gelatin medium and refrigerate it and it gels what does this mean?

answer

the organism is gelitinase negative because enzyme didn't break down the gelatin

question

starch hydrolysis

answer

- starch is broken down by AMYLASE enzyme
- starch agar medium is inoculated with organisms and incubated at 37C.
- the reagent iodine will then be added to test for the presence of starch, in the presence of starch iodine turns blueish-black.

POSITIVE FOR AMYLASE- agar surrounding colony does not turn bluish black after adding iodine (meaning starch is absent)

NEGATIVE FOR AMYLASE- agar surrounding the colony turns bluish black after adding iodine (starch is present meaning it wasn't broken down so it is amylase negative)

question

what breaks down starch/ hydrolyzes it

answer

amylase

question

what reagent is used to test for starch hydrolysis?

answer

iodine
(iodine turns starch blue/black, so if it does this then amylase was not present)

question

what turns starch blue/black and what test is it used for?

answer

iodine
starch hydrolysis via amylase

question

casein hydrolysis

answer

- the PROTEIN CASEIN is present in milk and is broken down by ENZYME CASEASE
- casein agar or milk agar is inoculated with organisms and incubated at 37C

CASEASE POSITIVE- clear zone surrounding colony, casein is hydrolyzed

CASEASE NEGATIVE- casein is intact, no breakdown via casease

question

what is casein and what media tests it

answer

a protein present in milk

a plate with casein will test for casein hydrolysis via casease

question

what breaks down casein

answer

casease

question

phenol red broth / carbohydrate formation

answer

-phenol red broth is used to determine an organisms ability to utilize a specific sugar
- medium contains pH indicator phenol red, the sugar of interest (glucose or lactose), protein and a small inverted tube called a durham tube
- at neutral pH, the tube will appear red
- organism is inoculated in tube for 24 hrs

SUGAR MAY BE METABOLIZED BY FERMENTATION- acids are produced which when released decrease the pH and cause the phenol red to turn yellow

GAS and METABOLIZED BY FERMENTATION- acid produced, so broth is YELLOW, but gas accumulation also appears as bubble in durham tube

SUGAR CANT BE METABOLIZED BY FERMENTATION- organism will use the protein present in the medium instead, and the by product of protein metabolism such as ammonia, increases the pH and as a result the phenol red turns BRIGHT PINK

REVERSION- if tubes are incubated for too long, then reversion can occur meaning the bacteria utilized all of the carbohydrate and then start breaking down the amino acids in the medium- causes pH to increase again and phenol red will revert from yellow to red on the surface of the medium
( HALF YELLOW HALF RED)

question

if phenol red broth/ carbohydrate formation test is done and tube is bright pink with no bubbles what does this mean?

answer

carbohydrate metabolism by fermentation did not take place, the organism used the protein in medium creating a by product such as ammonia which increased the pH and resulted in a BRIGHT PINK

question

if phenol red broth/ carbohydrate formation test is done and the tube is yellow with bubbles in tube what does this mean?

answer

sugar/carbs were metabolized by fermentation but gas was also produced as a by product of fermentation!!!

question

if phenol red broth/ carb formation test is done and the tube is yellow with no bubble what does this mean?

answer

the sugar was metabolized by fermentation, acids were produced lowering the pH and turning the phenol red YELLOW

question

if phono red broth is done and the top of the tube is red while the bottom is yellow what has happened?

answer

reversion
- tube was incubated too long
- bacteria utilize all carbohydrate and metabolize it via fermentation turning it YELLOW
- but then they begin to break down the amino acids in the medium also causing the pH to increase and turning the yellow phenol red BACK TO RED ON SURFACE

question

Triple sugar iron agar (TSI)

answer

- a differential medium that can differentiate gram negative enteric bacteria based on the ability to FERMENT certain CARBS to produce HYDROGEN SULFIDE!!!
- the medium contains (GLUCOSE, LACTOSE and SUCROSE), a pH indicator, iron, sulfur, and protein. the concentration of glucose is much lower than the other sugars.
- the organism is stabbed and streaked on the slant and incubated for exactly 24 hrs
- at neutral pH slant will be ORANGE RED
- results recorded are color reaction of the slant, color reaction of the butt, production of gas, and production of hydrogen sulfide gas

IF SUGAR IS METABOLZED- acid is produced that decreases pH to a YELLOW

IF GAS IS PRODUCED- crack seen in agar or agar may be pushed up the tube (PRODUCED AS BY PRODUCT OF METABOLIZING CARB)

IF SUGARS ARE NOT METABOLIZED- protein in the medium may be used by organism which causes pH to increase and change color to a MEDIUM MAGENTA OR BRIGHT RED

IF HYDROGEN SULFIDE GAS PRODUCED- it reacts with iron and forms a black precipitate

question

what are the three possible results of the TSI test

answer

1. sugar metabolized- acid produced, lowering pH, turns YELLOW. ***if gas is also produced CRACKS IN AGAR**

2. sugars not metabolized- protein in the medium used and pH increases turning medium MAGENTA OR BRIGHT RED

3. hydrogen sulfide gas produced- reacts with iron forming black precipitate

question

after you do the TSI test, a black precipitate has formed what does this mean?

answer

hydrogen sulfide gas was produced- iron reacts with it forming this black precipitate

question

after you do the TSI test, it is yellow and cracks are in the agar what does this mean?

answer

pH was lowered meaning the sugar was metabolized through fermentation and has turned YELLOW

the cracks indicate gas production

question

after doing TSI test, the tube is bright red, what does this mean?

answer

the sugar was not metabolized, so the medium protein was used instead, raising the pH causing RED COLOR

question

TSI 4 possibilities:

yellow butt and slant with gas:

yellow butt and red slant:

magenta butt and slant

magenta butt and slant with black precipitate

answer

yellow but and slant with gas: glucose plus one or two sugars metabolized, gas produced, no hydrogen sulfide

yellow butt and red slant: glucose only metabolized, no gas, no hydrogen sulfide

magenta butt and slant: no sugars metabolized, proteins metabolized, no gas and no or little hydrogen sulfide

magenta butt and slant with black precipitate: no sugars metabolized, proteins metabolized, no gas, sulfide production

question

IMViC test

answer

- used to identify enteric bacteria (gram negative facultative anaerobic bacteria that reside in GI tract) -INDOLE- indole is produced as a result of hydrolysis of tryptophan (an amino acid)- SIM (sulfur, indole, motility) media is a combined media that contains proteins, iron and sulfur-the sIM media is inoculated with the organism and KOVACS reagent is added after incubation. CHERRY RED- indole positive (tryptophan hydrolysis has occurred) NO RED- indole negative, no hydrolysis of tryptophan - SIM media can also detect release of hydrogen sulfide gas HYDROGEN SULFIDE PRODUCED- it reacts with iron and forms black precipitate Methyl red (M) and Voges-Proskauer tests (V) - all enteric organisms metabolize glucose to either acidic end products or neutral end products- the MRVP broth medium- contains glucose and other ingredients and can differentiate organisms that generate acidic and neutral end products- organism is inoculated into a single broth tube of MRVP. after incubation half is transferred into empty test tube so there are two tubes now. Methyl red test (M) - add drops of methyl red to one tube- if tube turns RED the end product is acidic (MR+)- if TUBE STAYS YELLOW- organism has not produced an acidic end product (MR-) voges proskaur test (V)- add drops of VP reagents A and B to other tube- RED/RED BROWN- organism has produced NEUTRAL end product VP+- YELLOW/YELLOW BROWN- VP-, the organism has not produced a neutral end product *** USUALLY MUTUALLY EXCLUSIVE- if an organism is MR+ then it is VP- citrate test- simmons media- inoculated and incubated- if medium turns BLUE- organism utilized citrate- if medium stays green- organism did not utilize citrate

question

IMViC is used for

answer

identifying enteric bacteria
(gram neg, facultative anaerobic bacteria in GI)

question

indole is produced as a result of

answer

hydrolysis of tryptophan (an amino acid)

question

SIM stands for

answer

sulfur, indole, motility
it contains proteins, iron, and sulfur

question

hydrogen sulfide in general is detected by

answer

reacting with iron and turning black precipitate

question

a positive indole test would be what

a negative indole test would be

the presence of indole means what

answer

SIM media turns cherry red!!!!!!!!

if negative- SIM media doesn't turn red

tryptophan hydrolysis has occurred

question

if SIM media is hydrogen sulfide positive what happens

answer

bottom of tube turns black

question

if SIM media is hydrogen sulfide negative what happens

answer

bottom of tube is yellow
(DOESNT TURN BLACK!)

question

the indole test (part of IMViC) uses what medium

answer

SIMS ( sulfur, indole, motility)

question

what is added to SIM media after inoculation and incubation?

answer

KOVAcS reagent

question

what medium is kovacs reagent used for

answer

SIM

question

what does SIM media detect

answer

indole production (hydrolysis of tryptophan)
release of hydrogen sulfide

question

the Methyl red (m) and votes proskauer tests (v) (PART OF IMViC) use what medium

answer

MRVP- which contain glucose and other ingredients and can differentiate organisms that generate acidic and neutral end products)

question

the MRVP test differentiates what

answer

enteric organisms that generate acidic or neural end products.

question

when using MRVP what tests are you doing

answer

MEthyl red (M) and Voges- proskauer (V) which are part of the IMViC

testing for neutral or acidic end products

question

when using the MRVP you do what after inoculation

answer

put half of tube in another test tube so you have 2 tubes

- one tube use for methyl red test, add drops of methyl red
IF IT TURNS RED- MR+ (produced acidic)

- one tube for votes proskauer test- add drops of VP reagents A and B
RED/ REDDISH BROWN- VP+ (neutral end product)

question

if during your voges proskauer test after adding ____________ reagent your tube turns RED what does this mean

answer

vp+ (neutral end products)

reagent- VP REAGENTS A AND B

question

if during you methyl red test if IMViC, you add methyl red and it turns RED this means what

answer

MR+ (acidic end product)

question

a neutral end product turns VP test what color

answer

VP+ turns tube RED or reddish brown
meaning a neutral end product was made

question

what reactions are usually mutually exclusive out of IMViC?

answer

methyl red and votes proskauer

if one is positive the other is negative

question

methyl red test turning red means neutral or acidic?

answer

acidic end product

question

VP test turning RED means neutral or acidic

answer

neutral end product

question

Citrate test uses what medium

answer

simmons citrate meidum
(contains citrate as sole source of carbon)

- organism inoculated and incubated
- if medium in the slant turns BLUE the citrate has been utilized
- if medium in slant STAYS GREEN- organism could not utilize citrate

question

negative citrate is what color

answer

green (simmons)

question

positive citrate test is what color

answer

blue (simmons)

question

urease test

answer

urease test -urea broth used to detect the presence of the enzyme urease, which hydrolyzes urea- as a result of hydrolysis, ammonia is released which increases pH.- urease is especially produced by organisms that reside in the stomach so they can survive in the acidic pH- YELLOWISH ORANGE- stayed this color after incubation, it is NEGATIVE FOR UREASE- BRIGHT PINK- organism is UREASE POSITIVE

question

urea broth is used for

answer

detecting the presence of urease WHICH IS FOUND IN ORGANISMS RESIDING IN STOMACH SO THEY CAN SURVIVE IN LOW ACIDIC pH

question

urea is produced by who

answer

enteric organisms ( produce it to fight acidic environment)

question

if tube stays yellowish orange after incubation in urea broth

answer

is is UREASE NEGATIVE

question

if tube turns bright pink after incubation in urea broth then

answer

IT IS UREASE POSITIVE

question

nitrate reduction test

answer

- nitrate (NO3) is a nutrient that can be utilized by bacteria and reduced to nitrite (NO2) which can be further deducted to nitrogen (N2) or nitrous oxide (N2O)
- the organism is inoculated in nitrate medium and incubated to determine if nitrate reduction (formation of NO2, N2 or N2O) occurred

STEPS
1. after incubation, nitrate reagents A and B are added to the broth which can detect the presence of nitrite (NO2)

RED BROTH- nitrite is present and this is POSITIVE
CLEAR- either the broth is still NO2 or it was reduced all the way to nitrogen (N2) or nitrous oxide (N2O)( NEEDS FURTHER TESTING)

step 2: if clear, zinc is added to tube
- if any original nitrate NO3 is present, zinc will convert it to nitrite(NO2). in this scenario tube will turn RED since nitrate reagents A and B (which detect nitrite) are already present from step 1 THIS IS NEGATIVE TEST FOR NITRATE REDUCTION since the organism did not reduce nitrate on its own

- if the tube STAYS CLEAR, then no original nitrate remained, meaning this is POSITIVE FOR NITRATE REDUCTION because N2 of N2O must have been present.

question

what is nitrate reduction

answer

the formation of NO2, N2 of N2O (from nitrate NO3)

question

if you do the nitrate reduction test and get a red tube initially this means

answer

nitrate (NO3) has been reduced to nitrite (NO2)

question

if you do the nitrate test and get a clear tube this means

answer

2 possibilities
1. still nitrate present, no reduction
2. nitrate has been reduced to nitrogen(N2) or nitrous oxide (N2O)

NEEDS FURTHER TESTING!!!!
ADD ZINC!!!!!

1. if zinc is added and it turns red- the zinc cause nitrate to convert to nitrite (NO2) meaning it is NEGATIVE FOR NITRATE REDUCTION since it didn't do it alone

2. if zinc is added and it stays clear, then no original nitrate was in tube meaning it was initially reduced to nitrogen or nitrous oxide so it is a POSITIVE RESULT FOR NITRATE REDUCTION

question

nitrate tubes

- yellow without zinc
- clear with zinc
- red with zinc

answer

yellow without zinc added- positive test, nitrate reduced to nitrite

clear with zinc- positive, nitrate reduced to nitrogen or nitrous oxide

red with zinc- negative for nitrate reduction, organism unable to reduce nitrate alone

question

what reagents are used for nitrate test

answer

nitrate reagent A and B
and ZINC

question

rapid identification methods

answer

multitest system- a signle unit that may run 10 or more tests at the same time
- results within 24 hours leading to an identification of the organism

ex: API20E, Minitek, Pathotec, enteropluri-test

question

enteropluri test

answer

allows for 15 biochemical determinations with one inoculation
- results read at 24 hours with a 5 digit code created based on the positive results
- the 5 digit code is then found on the code booklet giving you the identification of the organism

question

DNA fingerprinting

answer

- method of identification of an organism or individuals DNA
- it is useful for epidemiologists to determine the source of infection and identify the organism causing a particular infection
- once the DNA is collected it is cut into small pieces using restriction endonucleases enzymes
- these enzymes cut DNA at specific sequence called the restriction site
- DNA samples with different nucleotide sequences such as samples of different bacteria will be cut differently by the same enzyme and yield different sizes of fragments

question

what is DNA fingerprint useful for

answer

epidemiologists
DETERMINING SOURCE OF INFECTION AND IDENTIFYING ORGANISM CAUSING IT

question

DNA is cut into small pieces during DNA fingerprinting by what?

answer

restriction endonucleases (enzymes)

question

how are DNA fingerprinting/dna fragments examined?

answer

gel electrophoresis
1. restriction enzymes cleave DNA into smaller segments of various sizes
2. DNA segments are loaded into wells in a porous gel, the gel floats in a buffer solution within a chamber between two electrodes
3. when an electric current is passed through the chamber DNA fragments move toward the positively charged cathode
4. smaller DNA segments move faster and father than larger creating the difference in appearance

question

how to do gel electrophoresis

answer

- DNA samples are mixed with a loading dye and loaded into the wells of an agarose gel
- an electric field is applied across the gel causing the negatively charged DNA fragments to move from their origin (sample well) through the gel to the positive end
- the gel matrix acts as a sieve through which smaller fragements move faster than large ones
- a pattern of bands are produced fro each sample and are made visible by staining with a dye (ethidium bromide) that binds to the DNA molecule
- the bands show up under a UV lamp

question

passive agglutination

answer

- agglutination happens when antibodies bind to particulate antigens or cells such as bacteria and form visible climbs called agglutinates
- all diagnostic reactions use something to make antigen- antibody reactions detectable
- passive agglutination utilize beads attached to either antigen or antibodies. when the corresponding antibody or antigen from the patient sample is added clumping will be observed.

question

what does passive agglutination utilize to make antibody antigen reactions detectable

answer

beads attached to either antigens or antibodies

**when the corresponding antibody or antigen from the patient sample is added, then visible clumping will be observed

LAB FINAL MICROBIOLOGY!!!!!! – Flashcards

Unlock all answers in this set

Haven't found what you were looking for?

Search for samples, answers to your questions and flashcards