Gen MICROBIO LAB PRACTICAL studyguide Cartes

Steps of the Gram-stain and color of G (+) and (-) along the way

1. smear

- gram (+) control, unknown and gram (-)

- add 1 loop of water to each oval drawn on slide with wax pencil

- use needle to mix a small sample of culture into the appropriate water drop and spread over whole area

2. heat fix sample

3. flood each smeat with CRYSTAL VIOLET (1min) & wash with water

- G(+): purple

G (-): purple

4. flood smears with IODINE (1 min) & wash w/ tap water

- G(+): purple

G (-): purple

5. Decolorize: wash each smear with decolorizer until no purple color in draining liquid

- G(+): purple

G (-): colorless

(USED ALCOHOL-ACETONE)

6. briefly wash smear with tap water

7. counterstain bacteria with SAFRANIN

- G(+): purple

G (-): pink

8. wash with tap water

9. examine slides under bright-field. NO COVER SLIP

WHAT ARE some common methods of visualization for metabolic tets?

- pH indicator ( color change in medium)

- inherently visible reaction product, (can be observed without add indictors or chemicals)

- add chem compound after organism has grown ( chem cpd add to reaction will modify the product or intermediate to form a colored cpd)

what is an enzyme

biological catalysts that lower energy of activation for biochem reactions and permit essential metabolic steps to proceed rapidly

NOT consumed in the reaction

Micrococcus leteus

G (+)

catalase positive

Streptococcus thermophile

G (+)

catalase negative

what is the rate of a enzyme-catalyzed raction depended on

temperature

concentration of enzyme

concentration of substrate

affinity of enzyme for the substrate

why are enzimatic tests good for determining the identity of an organism?

because a microbe's physiological properties are determined by its enzymes capacities

also a microorganism habitat and its interactions with other organisms are a direct reflection of the organism's enzyme complement

What is catalaze? what is its purpose?

catalaze is an enzyme that catalyses the breakdown of hydrogen peroxide (H2O2 - a toxic by-product of aerobic respiration metabolism) to water and gaseous oxygen

purpose of enzymatic test CATALASE? Results

done for GRAM POSITIVE organisms only

to determine if a mciroorganism has catalase

the presence of catalase indicates that the organism MOST LIKELY performes aerobic respiration

add drop of hydrogen peroxide to loopful of bacteria spread on microscope slide. if bubbles of oxygen appear, POSITIVE for catalase

what type of organism can CAtalase enzymatic test be done in?

Gram POSITIVE only

Procedure of CATALASE enzymatic test

add drop of hydrogen peroxide to a loop full of bacteria spread on microscope slide. observe if bubbles or no

what is the purpose of the enzymatic test OXIDASE?

Only done in G (-)

common test to differentiate between families of Pseudomonadaceae (oxidase positive) and Enterobacteriaceae (oxidase negative)

test used to learn if an organism produces certian cytochrome c oxidase.

cytochrome c oxidase is the terminal link in an electron transport chain where oxygen in the terminal electron acceptor.

cytochrome c oxidase is a lipoprotein complex that transfers electrons from cytochrome c to oxygen and pumps protons (H+) across the cytoplasmic membrane

what type of organism can OXIDASE enzymatic test be done in?

G (-) ONLY

Procedure of OXIDASE enzymatic test

1. get amount of culture on a swab

2. use dropper bottle, add drops of 1% tetramethyl-p-phenylenediamine dihydrochloride

3. turn purple ater 10 seconds, oxidase POSITIVE

RESULTS of OXIDASE enzymatic test

turn purple after 10 seconds, oxidase positive

- yes cytochrome oxidase, most likely Pseudomonadaceae family

no purple after 10 seconds, oxidase negative

- no cytochrome oxidase, most likely Enterobacteriaceae family

reagent will turn purple after 20-30 seconds b/c oxidation by the air -> FALSE positive

metabolic test done on G (-) organimss ONLY

CITRATE utilization

MR-VP (Methyl Red & Voges-Proskauer)

SIM (Sulfide Indole Motility)

Nitrate reduction

Eosin-methylene

metabolic tests for G (+) ONLY

Bile Esculine slant

Mannitol salt

6.5% salt broth

Muller Hinton agar and Novobiocin

metabolic tests for G (+) & G (-) Unknowns

carbohydrate utilization

oxygen tolerance

purpose of the CITRATE UTILIZATION metabolic test

G (-) ONLY

to evaluate the microorganisms ability to utilise citrate as its sole source of carbon

negative result: green

positive result: royal blue

Simmons citrate agar (used for CITRATE UTILIZATION metabolic test (G -)) components

sodium citrate - Carbon source

ammonium salts - nitrogen source

bromthymol blue - pH indicator (growth makes pH rise, pH indicator turns green-> blue)

Citrate slant

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (-)

Original color: GREEN

Negative result: GREEN

Positive result: ROYAL BLUE

Any chem add to visualize reaction:

METHYL RED TEST (MR) metabolic test. purpose

G (-) ONLY

organism's primary fermentation pathway = mixed acid fermentation

(produce - acetic, formic, lactic, and succinic acid + ethanol, molecular H, and carbon dioxide -> accumulation of acids, drop pH. Methyl red bellow pH 4.8 = red )

Methyl Red media

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G(-)

Original color: FAINT YELLOW

Negative result: YELLOW

Positive result: RED

Any chem add to visualize reaction: METHYL RED INDICATOR

Voges-Proskaur (VP) test PURPOSE

G (-)

Microorganisms that do butanediol fermentation pathway

the test detects an intermediary metabolite, Acetoin

VP (Voges-Proskauer)

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (-)

Original color: FAINT YELLOW

Negative result: BROWN COLOR

Positive result: RED/BURGUNDY

Any chem add to visualize reaction: REAGENT 1: 40% KOH

REAGENT 2: ALPHA-NAPTHOL

SIM metabolic test purpose

G (-)

SIM - Sulfide Indole Motility

produce hydrogen sulfide? (H2S

form indole?

motility?

SIM

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (-)

Original color: PALE YELLOW

Negative result:

- sulfur: NO COLOR CHANGE

- indole: NO COLOR CHANGE

- motility: GROWTH ONLY ON CENTRAL STAB LINE

Positive result:

- sulfur: BLACK PRECIPITATE

- indole: PINK/DEEP MAGENTA AT TOP OF MEDIUM

- motility: GROWTH RADAITE FROM CENTRAL STAB LINE OUR

Any chem add to visualize reaction: KOVAC'S REAGENT add to detect presence of INDOLE

NITRATE REDUCTION metabolic test purpose

G (-)

microorganism have enzymes nitrate (NO3) reductase and nitrite (NO2) reductase?

Test used to determine if organism reduces nitrate (NO3) to nitrite (NO2)

& if ability of nitrification on both nitrate and nitrite to produce molecular nitrogen

Nitrate reduction

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (-)

Original color: PALE YELLOW

Negative result:

- NO BUBBLES, NO COLOR CHANGE AFTER ADD REAGENT A& B, RED COLOR AFTER ADD Zn POWDER -> NO NITRATE REDUCTION

Positive result:

- LARGE BUBBLE IN DURHAM TUBE-> NITRATE AND NITRITE REDUCTASES (NITRATES -> NITRITES -> NITROGEN (g))

- NO BUBBLE, RED AFTER ADD REAGEN A & B, POSITIVE NITRATE REDUCTION TO NITRITE

Any chem add to visualize reaction: REAGENT A: SULFURIC ACID

REAGENT B: ALPHA-NAPHTHYLAMINE

If no color change after add reagents, Zn powder add

In a Nitrate reduction metabolic test, what is the purpose of adding Zn powder when there was no color change after adding the reagents A & B?

To confirm a true negative result.

the addition of Zn powder forces the reduction of any remaining NITRATE to nitrITE

If the medium turns red after adding zinc, it confirms that nitrATE was still present in the medium, meaning the bacteria DID NOT reduce nitrate

possible outcomes for Eosin-Methylene Blue (EMB) petri dish

G (-)

Blue-black with GREEN METALLIC SHEEN = POSITIVE (microorganism ferment lactose rapidly)

colorless, mucoid - non-lactose fermenter (NEGATIVE)

Purpose of the Eosin-Methylene Blue EMB metabolic test

G (-)

the medium slighly selecive for G (-), it allows us to differentiate btw organims that can FERMENT LACTOSE and those that cannot

purpose of Bile Esculin slant

G (+)

Selective and differential medium

selects for bile tolerant bacteria and differentiate btw bacteria that can or cannot convert esculin to esculitin

BILE ESCULIN SLANT

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (+)

Original color: CLEAR, AMBER

Negative result: CLEAR AMBER

Positive result: BLACK

Any chem add to visualize reaction:

Mannitol salt metabolic test

G (+)

Selective and differential medium

selects for halotolerant organisms

differential - organisms can ferment mannitol, which cannot

possible outcomes Mannitol salt slant agar

G (+)

NEGATIVE: No growth, no change in color

POSITIVE:

- POSITIVE mannitol fermentation & halotolerance = Growth and yellow agar

- POSTIVE halotolerance, NEGATIVE mannitol ferment= pink agar

6.5 % salt broth test purpose

G (+)

determine if organisms is halotolerant

6.5 % NaCl TSB

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (+)

Original color: LIGHT AMBER

Negative result: CLEAR YELLOW

Positive result: TURBID

Any chem add to visualize reaction:

Novobiocin sensitivity test purpose

G (+)

Test for antimicrobial susceptibility testing

medium used: Mueller Hinton Agar (all - purpose)

add Novobiocin disc

Novobiocin is antimicrobial agent target DNA gyrase of bacteria

what is Novobiocin

an antimicrobial agent that targets DNA gyrase of bacteri. interfer with DNA replication

possible outcomes Novobiocin sensitivity test

G (+)

NO ZONE OF INHIBITION (growth througout) - organism is RESISTANT to Novobiocin

ZONE PRESENT, diameter larger than 16mm, organism sensitive to Novobiocin

purpose of carbohydrate utilization (glucose OR lactose)

G (+) & G (-)

to determine if microorganism can ferment

- GLUCOSE by detecting end products (acid or hydrogen gas)

- LACTOSE by detecting acidic end product

Phenol red borth with GLUCOSE

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (+) & G (-)

Original color: RED

Negative result: NO COLOR CHANGE

Positive result: YELLOW, Bubbles in Durham tube

Any chem add to visualize reaction:

Phenol red browth with LACTOSE

Original color:

Negative result:

Positive result:

Any chem add to visualize reaction:

G (+) & G (-)

Original color: RED

Negative result: NO CHANGE COLOR

Positive result: YELLOW

Any chem add to visualize reaction:

Purpose of the FLUID THIOGLYCOLLATE MEDIUM

to determine the relationship between microorganisms and oxygen concentration

possible outcomes for Fluid Thioglycollate medium metabolic test

OBLIGATE AEROBE: in oxic zone

OBLIGATE ANAEROBE: In bottom of anoxic zone

FACULTATIVE ANAEROBE: Throughtout all tube, high concentration in oxic zone

MICROAEROPHILLIC: Some in oxic zone. Highest concentration in anoxic zone but RIGHT at boarder with oxic zone

AEROTOLERANT: Evenly spread through out all zones

obligate aerobe

organism that requires oxygen in order to live

obligate anaerobe

organism that cannot live in the presence of oxygen

facultative anaerobe

organism that can survive with or without oxygen

if oxygen present, prefers these conditions

microaerophillic

requires only a small amount of oxygen

likes oxygen levels right bellow atmospheric levels

Aerotolerant

do not utilize oxygen but can survive and grow in its presence

in a gram stain, wHAT is the purpose of adding a counterstain? what are the colors of G (+) AND G(-)?

a counterstain is used to recolor cells that have lost the primary stain after alcohol treatment. It contrasts with the primary stain

in lab we used SAFRIN

- G(+): purple

G (-): pink

DURING A GRAM stain what is the purpose of decolorizing agent? And what are the colors of G (+) and G (-) after wash?

it is a solvent, acetone, alcohol or a mix that removes the primary stain from the Gram-negative cell.

The outer membrane dissolves and the G (-) appears colorless.

Also "dehydrates" the G (+) cell wall. the peptidoglycan mesh shrinks and traps crystain violete-iodine complexes.

In both bacterial cells, the plasma membrane is disrupted

in lab we used ALCOHOL-ACETONE

- G(+): purple

G (-): colorless

in a gram stain, what is the purpose of washing the stains with iodine? what are the colors of G (+) and G (-) after the wash?

Iodine is as a mordant so it fixes the crystal violet stain so that it resists washing out.

This step enhanses the color by enhancing a complex formation between the dye and the bacterial cell

- G(+): purple

G (-): purple

During gram stain, waht is the purpose of a primary stain?

what is the color of a gram (+) and gram (-) organims after staining it ?

primary stains stain all cells purple.

in lab we used CRYSTAL VIOLET

G(+): purple

G (-): purple

what type of stain is CRYSTAL VIOLET? what is it used for?

BASIC dye (positive charge)

used for Gram stain

stain cells purple

what other dye could be used as primary stain for gram stain?

basic dyes

ex. methaline blue

gram stain colors = diff

in a gram stain, what is the purpose of heat fixing

to permanently adhere cells into the slide.

when polysaccharides are exposed to heat, they denaturalize so need to make sure not heat up the slide too much or else the gram stain will be unsuccesfull.

In a gram stain, what is the purpose of always smearing a positive and negtive control?

allows us to evaluate our technique and compare the results with the unknown so we cna determine the Gram reaction of it.

Staphylococci

bacteria that grow in clusters that resemble a bunch of grapes.

Streptobacilli

rod-shaped bacteria occurring in chains

streptococci

bacteria that form a chain

diplococci

Spherical bacteria that grow in pairs

tetrads

4 cocci in a square

sarcinal

cuboidal structures of eight or more cells

coccibacilli

very short rods

vibrio

comma shaped

spirilla

spiral, noodle shaped bacteria

Spirochetes

spiral-shaped bacteria that have flexible walls and are capable of movement

what is Negative stain? what is its purpose?

a simple stain that stains the background but leaves the bacteria unstained.

use NIGROSIN (ACIDIC DYE)

use live cells

Purpose: determine an organisms cellular morphology

WHAt is the purpose of gram stain ? what is the significance?

To distinguish between Gram-positive and Gram-negative bacteria.

gram (+) bacteria has a thick peptidoglycan layer in cell wall

gram (-) has a thin peptidoglycan layer. it deos have outer membrane that is composed of lipid bilayer with embeded preoteins and other macromolecules

what type of dye is NIGROSIN and what is it usually used for?

acedic dye

used for negative stain

what is the significance of gram stain procedure in mdeciine?

helps determine what type of drug to utilize to combat the infection by the bacteria

POLYMYXIN for G (-)

isrupt lipid membranes in outer membrane

PENICILLIN for G (+)

interfeer with cell wall construction, prevnt peptidoglycan from being assembled

what are the results of a gram stain?

Gram positive will be purple

Gram negative will be pink

rESTULT of gram stain : G (+) looked pink

what does this mean?

probably over decolorized samples, shrunk cell wall of G + peptidoglycan to a level, inhibiting its ability to retain CRYSTAL violet die

the process for identifying an unknown microbe

from mix culture with unknown ->

unknown isolation via QSP and Slant transfer ->

wet mount so anaylze morphology using miroscopy->

gram stain (determine if gram + or gram - ->

biochemical tests (enzymatic and emtabolic tests) ->

compare to database

In micropipets what does a number in a diffeernt color or under a line mean?

usually it denotes the tenth place

In drawings for microscoscope pciture, What are the things EVERY picture should include?

1. total magnification

( occular lense times 10)

(write as # X)

2. Name of specimen

3. type of microscopy

BF = bright field

PC = phase contrast

4. description of morphology and motility

5. scale bar

( magnification 100x (10 * 10) = 10

400x = 2.5

1000x = 1.0 )

scale double the size of biggest bacteria

filamentous form

irregular form

punctiform

tiny colonies

bacterial colonies surface is usually...

shiny, smooth

tesxture of bacteria growth can be described as

dry

moist

mucoid

brittle

viscous

butryrous (buttery)

important descriptive terms of the color of bacterial colonies

opawue, cloudy, translucent, iridescent (show lumunois color, change when seen from diff angles)

elevation of colonies we observed in class were usually

flat

Description morphology of bacterail colonies

what is the combination of type of microscopy with correct objective lense (magnification)

BF 0

PC 1 with 10x

PC 2 with 40x

PC 3 100x

How to define the scale bar for a microscopy picture/drawing?

for a magnification of 100x (10 * 10) = 10

400x = 2.5

1000x = 1.0

scale double the size of biggest bacteria

how to determine the total magnification

occular lense times 10

(write as # X)

description of motility

rum & tumble (flagella movement)

swarming ( move across entiere plate as group)

what do microorganisms need to survive?

carbon source, nitrogen, phosphorous and other minerals

Suitable temperature and pH (buffers, weak acids, or bases)

water nutrient transport

and oxygen (or lack of it)

What kind of medium is Trypcase Soy Broth (TSB). What are its ingridients and theri purpose?

pacreatic digest of casein -

enzymatic digest of soybean meal -

NaCl -

Dextrose -

Dipotassium phosphate -

General, all purpose medium

pacreatic digest of casein -

enzymatic digest of soybean meal - nutrients

NaCl - osmotic presure

Dextrose - carbon source

Dipotassium phosphate - buffer

pH 7.3 @ 25C

What kind of medium is Trypcase Soy Agar (TSA). What are its ingridients and theri purpose?

tryptone

soytone

NaCl -

Agar

General all-purpose agar

tryptone (pancreatic digest of casein)

soytone (papaic digest of soybean meal)

NaCl - osmotic pressure

Agar

What kind of medium is Mycophil. What are its ingridients and their purpose?

papaic digest of soybean meal

dextrose -

agar

final pH

selective medium by pH for fungi

papaic digest of soybean meal

dextrose - carbon source

agar

final pH 4.7 at 25C

What kind of medium is MacConkey Agar. What are its ingridients and their purpose?

a selective and differential agar.

Selective for G (-)

selective factor is ampicillin

if microorganism has gene for ampicillin resistant, will grow

differential factor is bacto lactose

if microorganism can digest lactose, agar will be maroon (pH go down)

if cant digest lactose, agar = amber color and colorless colonies (pH go up)

What color do colonies turn in a MacConkey medium if they CANNOT metabolize lactose?

agar turns ambar color and colonies are colorless because to survive, organisms aminate proteins causing the pH go UP

What color do colonies turn in a MacConkey medium if they CAN metabolize lactose?

colonies turn a maroon color because they ferment lactose and produce acid, making the pH go DOWN

what is the purpose of petone, which is found in many agar?

peptone is the principal source of organic nitrogen and may contain carbohydrates or vitamins

% w/v

g/100 mL

what is a medium

sterile base that has all nutrients required for microbial culture of cells to grow