MIC 206 Practical 2 study guide :)

Tips on finding stuff on microscope (5)

- clean microscope with lens paper only (not bibulous paper) - find a reference point (circle or clumps) - start on low power (10x) and move the stage - switch to the high power (40x) only move fine adjustment - switch to oil power (100x) (only move fine adjustment

Oxidase Production Test: Cytochrome C Oxidase

- cytochrome c is a highly conserved protein across the spectrum of complex species, found in plants, animals, and many unicellular organisms - allows one to study the evolutionary relationships of organisms

5 examples of Biochemical Characteristics used for identification

- degradation enzyme - sugar fermentation - citrate as sole carbon source - malonate as sole carbon source - type of fermentation end products

Gram-Positive bacteria (2)

- have thick, mesh-like layer of peptidoglycan - contain up to 100 sheets of peptidoglycan stacked upon one another

Growth Considerations: What type of media does one use? (2)

- nutrient factors: vitamins, minerals, energy, carbon - selective, differential, complex, define

Results of Biochemical RXN (2)

- positive and negative results are equal - test importance depends on how the resource is organized

when MR (-) then VP is

(+) and vice versa

Catalase Production Test: Positive and Negative Result

+ = bubbles - = no bubbles

Identification Biochemical Characteristics #5: Type of fermentation end products (2)

- Methyl red (MR) - Vogus Proskauer (VP)

6 stains that belong to Physical Identification Groups

- simple stain - shape - gram stain (peptidoglycan) - capsule stain (capsule) - endospore stain (endospore former) - acid fast stain (mycolic acid)

Phenol Red Basics: Fermentation (3)

- using carbohydrate (sugar) converting it to acids or gas (h2 and CO2) - the release of acid will lower the pH of the media - to perform fermentation glycolysis is used (multiple enzymes) in this process

unknown pathogenic bacteria growth considerations (4.5)

- what type of media does one use? (MEDIA) - what should be the incubation temp? (TEMP) - what should be the oxygen conditions for incubation? (OXYGEN) - others: pH, salt...

2 simple steps in identifying Unknown Pathogenic Bacteria

-1. grow each of the colonies as a pure culture (swab -> quadrant streak) 2. now cultures can be studied to identify genus and species

Capsule (3)

-Water-soluble, preventing dye from adhering to them: composed of polysaccharides/ polypeptides. -Increase virulence by protecting the bacteria against phagocytosis and antibodies. -Helps the bacteria attach to surfaces.

Gram-Negative bacteria (3)

have a thin layer of peptidoglycan and an outer membrane - only has min of 10 sheets of peptidoglycan (10%) -made up of mainly outer membrane

Function of Mycolic acid w/ examples (2)

helps protect against certain antibiotics/ antimicrobials ex: genus "mycobacterium"

Phenylalanine Deaminase Test (2)

identifies organisms that can synthesize the enzyme phenylalanine deaminase -Phenylalanase remove's phenylalanine amine group to form phenylpyruvic acid and NH2

Phenol Red Test

identify what types of carbohydrate a bacteria can ferment or use and the types of end products

Catalase Production Test: Input and Output

input: hydrogen peroxide output: oxygen + water

Mixed Acid Fermentation: Methyl Red Test: Glucose (2)

is converted to organic or mixed acids - multiple enzymes are involved in the conversion

Chromophores

stains contain color bearing ions

Negative (capsule) stain

stains the background which surrounds the unstained microbe

Nitrate Reduction Test

tests whether an organism is capable of reducing NO3 to NO2 via Nitrate Reductase

examples of endospore diseases (5) *NAME AT LEAST 2

tetanus anthrax botulism gas gangrene pseudomembranous colitis

Why do Gram-Positive bacteria have more peptidoglycan?

they are trying to withstand higher internal pressure

Nitrate Reduction Test: Role of Zinc Dust (4)

used to confirm negative result - the dust will reduce NO3 --> NO2 red: confirms negative result clear: other reduced nitrogen compounds

Oxidase Production Test

used to determine if Cytochrome C Oxidase is a member of the electron transport chain of the organism

Phenol Red Basics: growth from proteins

when bacteria use proteins for growth this will result in the release of ammonia (NH3) and an increase in pH of the media

Simmons Citrate Utilization Test

Determines the ability of an organism to use Sodium Citrate as its sole carbon source

Simmons Citrate Utilization Test: Negative Result (2)

green organism cannot utilize citrate

color of endospore

green (malachite green)

How do you know you are looking at a capsule stain?

purple cells surrounded by a clear, colorless zone (capsule) on dark background

Oxidase Production Test: Positive Result

purple/blue oxidase reagent will donate its electrons to cytochrome c oxidase

Nitrate Reduction Test: Positive and Negative Result

red= positive for nitrate/nitrite clear= negative

Color of acid-fast cells

reddish/ pink -whatever color used as carbolfuchsion stain

What do Deamination Reactions do?

remove amine (N) groups producing acidic compounds

Decolorizer

removes primary Stain from Gram-Negative cells

Growth Considerations: What should be the incubation temp? (3)

room 37 C, 25 C, or body temp

Peptidoglycan (2)

-cell wall component unique to bacteria -largely responsible for the strength of the cell wall

Big Picture (3)

-organisms are different from each other based on the genes they posses -genes code for unique traits and abilities (enzymes and proteins) - biochemical tests allow researcher to differentiate organisms based on their traits

Counter Stain (3)

-second dye -added after the decolorizer is washed out -determines color of Gram-Negative cells

Miniaturized Test System

-used in health care systems -they can inoculate many tests simultaneously and incubate the whole test system (cost $100 per person)

Simple Stain (6 steps)

1. Smear Prep 2. Heat fix 3. Flood Slide with Stain 4. Wash slide with Water 5. Use bibulous paper and blot dry (gently) 6. Record images under 100x magnification (oil objective lens)

7 steps of Endospore Staining

1. Smear prep and Heat Fix 2. Apply malachite green continuously over steam for 10 minutes 3. Rinse with water 4. Counterstain with Safranin for 1 minute 5. Rinse with water 6. Gently blot with bibulous paper 7. view under oil immersion lens (100x)

9 steps of Capsule Stain (2 slides)

1. apply line of bacteria to one end of slide 2. apply one drop of Nigrosin to that same end of slide 3. take a clean slide, place it on the surface of the first slide at the drop site 4. hold clean slide at 45 degree angle and drag it to the other side of the slide to spread the stain and bacteria throughout 5. air dry, NO HEAT FIX 6. flood slide with Crystal Violet 7. wash with water 8. gently blot with absorbent paper 9. view with oil objective (100x)

Smear Prep (5 Steps)

1. draw a circle on the bottom side of the slide 2. Broth: one or two loops of bacteria Plate: add water to slide then add colony from plate 3. smear it around the slide (in the circle) 4. allow it to air dry 5. HEAT FIX: hold slide in front of the Bacinerator for 5 secs

Why are endospores resistant? (2)

1. have tough protein coats highly resistant to normal staining procedures. 2. impermeable spore coat (exosporium) allows for resistance to unfavorable conditions

10 steps of Gram Stain

1. smear and heat fix 2. flood side with crystal violet (primary stain) for 60 secs 3. wash with water 4. flood with grams iodine (mordant) for 60 sec 5. wash with water 6. decolorize with 95% ethanol (decolorizing agent) for 2-3 sec 7. wash with water 8. flood with safranin (counter stain) for 60 secs 9. wash with water 10. blot with absorbent paper

9 steps of Acid Fast Stain/ Siehl-Neelsen (steam)

1. smear prep and heat fix 2. flood slide with carbolfuchsion (primary stain) for 8 minutes over steam 3. wash with water 4. decolorize with acid alcohol (decolorizing agent) (acid fast cell resist decolorization) 5. Wash with water 6. Flood with methylene blue (counter stain) for 60 seconds 7. wash with water 8. blot with absorbent paper 9. view with oil objective lens (100x)

Nitrate Reduction Test: Reagents used to test for NO2

9 drops of Nitrate solution A (sulfanilic acid) 9 drops of Nitrate solution B (dimethyl-alpha-naphthalamine)

Gram staining allows one to differentiate

90% of bacterial species into two large groups (gram positive and gram negative) based on the chemical and physical properties of their cell walls (peptidoglycan)

Phenol Red Test: Results (4)

A/G: fermentation and gas production (yellow with bubble) A/-: fermentation, no gas production (yellow no bubble) -/-: no fermentation and no gas production (orange) k: deamination of proteins, amino acids produces ammonia (NH3) (red)

acidic stains w/ example (1)

ANIONS (- charge) Nigrosin

Heat Fix

Adheres bacterial cells to the slide and prevents loss of sample during staining procedure

Starch Hydrolysis: Enzyme

Amylase will break down starch (amylose: long chains of glucose) into individual glucose units

Simmons Citrate Utilization Test: pH indicator (3)

Bromothymol Blue initial pH of 6.9 - green pH of 7.6 or more - deep blue

Basic/Alkaline Stains w/ examples (3)

CATIONS (+ charged) Methylene blue, Safranin & Crystal violet.

Simmons Citrate Utilization Test: Enzyme

CITRATE PERMEASE required to transport sodium citrate across the cell membrane

Catalase Production Test: Enzyme

Catalase

Catalase Production Test: Reagents

Hydrogen Peroxide

Mixed Acid Fermentation: Methyl Red Test

MR test identifies organisms that produce stable acid end products

Methyl Red and Voges-Proskauer (2)

MRVP broth is used for both MR and VP tests -tests for multiple reactions

Identification Biochemical Characteristics #2: Sugar Fermentation (3)

Mannitol, Lactose, Glucose

nitrate reduction/ nitrate broth equation

NO3 --> No2 --> N2

Phenol Red Media Composition of each tube (4 in ea. of the 4 tubes)

PR Base: no carbohydrate, phenol red, protein, and durham tube PR Glucose: glucose (carbohydrate), phenol red, protein, and durham tube PR Lactose: lactose (carbohydrate), phenol red, protein, and durham tube PR Sucrose: sucrose (carbohydrate), phenol red, protein, and durham tube

Phenol Red Basics: pH indicator (4)

Phenol Red - pink: at alkaline (>7.4) pH - red at neutral (6.9 - 7.3) pH - yellow at acidic (<6.8) pH

What is mycolic acid composed of? (3)

fatty acids, waxes, and complex lipids that make up the cell walls

Phenylalanine Deaminase Test: Reagent

add 5 drops of 10% FeCI3 -FeCl3 reacts with the phenylpyruvic acid to turn the slant avocado green

H2O2 is toxic to enzyme systems of microbes but is generally produced by

aerobic and facultative microbes as a byproduct of aerobic metabolism

Growth Considerations: What should be the oxygen conditions for incubation? (3)

aerobic, anaerobic, microaerophilic environment

Phenol Red Basics: Durham tube (2)

are invented small test tubes used to trap gas that would be produced during fermentation -bubble indicates gas production

Primary Stain

first stain added after heat-fixing -color of Gram Positive cells

What are the 3 shapes of cell morphology

bacillus (rod) cocci spirillum or curved

Starch Hydrolysis

bacteria's ability to hydrolyze starch with amylase

Color of nonacid-fast cells

blue

Simmons Citrate Utilization Test: Positive Result (3)

blue -organsims utilizes citrate permease -medium becomes more basic and pH indicator turns blue

Malonate Test: Positive and Negative Result

blue + Green -

Identification Biochemical Characteristics #1: Degradation Enzyme (6)

catalase nitrate reductase urease tryptophanase lipase phenylalanine deaminase

What 2 things can be identified from a Simple Stain?

cell morphology and arrangement

Oxidase Production Test: Negative result

clear or no reaction no cytochrome c oxidase

By adding colored compounds, we add ______ for visability

contrast

What stains do vegetative cells readily take up?

counter stain (SAFRANIN)

2, 3 Butanediol Fermentation: Voges Proskauer Test

detects acetoin, which is a precursor of 2,3-butanediol -they are always found together

Locations of endospores

different locations within the cell

endospores

dormant life forms

Mixed Acid Fermentation: Methyl Red Test: Test for Presence of Mixed Acids and Gases (4)

lactate, acetate, succinate, formate

Why does malachite green rinse easily from the vegetative cells? (2)

malachite green is water-soluble and does not adhere well to the cell -since the vegetative cells have been disrupted by heat, the stains rinses easily from cells

Malonate Test

malonate broth tests an organisms ability to utilize Malonate as a sole carbon source

Identification of Physical Characteristics by Staining: why are physical characteristics not useful with when identifying a specific microorganisms?

many organisms are similar (many are gram negative or rod shaped)

Mixed Acid Fermentation: Methyl Red Test: pH indicator (4)

methyl red pH indicator - pH less than 4.4 is red - pH 4.4-6.2 is orange - pH greater than 7.2 is yellow

bacterial endospore is not the same as

mold spore

Acid-fast bacteria unique cell wall contains

mycolic acid

What are the charge of a bacterial cell?

negative

Nigrosin (3)

negatively charged and is repelled by the negative charge of the bacteria (stains background) -ACIDIC dye -NEG cause it gives up hydrogen ion

Catalase Production Test: Principle of test

organisms with catalase can render hydrogen peroxide harmless

Malonate Test: pH indicator

pH less than 6.9: Green pH greater than 7.6: Blue

Why is heat/steam is applied to the primary stain of an Endospore Stain?

penetrate the endospore

color of vegetative cells

pink (safranin)

Identification of Physical Characteristics by Staining: Shape and Gram reactions allow a researcher to

place the unknown organism into a group

crystal violet (2)

positively charged and stains the bacterial cells - BASIC dye