MCB 3020 Lab Midterm Stuff

Lab attire/PPE

Includes lab coat, gloves, goggles, and long pants

70% EtOH

This is what you clean your lab bench with BEFORE each lab

Bleach wipes

This is what you clean your lab bench with AFTER each lab

Inoculating loop

Used to transfer a culture of microorganisms to an agar medium in a petri dish, to an agar slant, or to another liquid medium

Microincinerator

Used to sterilize inoculating loops and test tube openings

Aseptic technique

A procedure performed under sterile conditions; used to limit the presence and spread of potentially harmful organisms

Two Tube Transfer

Method in which aseptic technique is used to transfer a sample from a bacterial culture to another uninoculated medium

Cationic dyes

Basic dyes, positively charged chromophores

Anionic dyes

Acidic dyes, negatively charged chromophores

Negative stain

Type of stain that colors the background, not the cell itself; cells are not heat fixed

Simple stain

Type of stain that uses one dye to stain all cells present; colors cell surface

Illumination

Light source

Condensor

Direct light towards objective lens in bright field microscopy

Iris diaphragm

Adjusts diameter of cone of light so that it just fills objective lens

Total magnification

Ocular lens x objective lens

Resolution

Smallest distance between two objects which can be seen as separate

Oil immersion

Must be done for 100X objective lens

Glass

Oil has the same refractive index as _________.

Differential stain

Detects differences between organisms through the use of many dyes and reagants

Structural stain

Confirms structural characteristics of cells

Gram stain

A type of differential stain that differentiates bacteria based on the amount of peptidoglycan

Gram's iodine

Mordant in a Gram-stain

95% EtOH

Decolorizer in Gram-stain

Crystal violet- Gram's iodine- 95% Ethanol- Safranin

This is the order for a Gram-stain

Gram-positive

These stain purple on a Gram-stain.

Gram-negative

These stain pink on a Gram-stain.

Acid-fast stain

A type of differential stain that is used for microorganisms with high mycolic acid content

Ziehl-Neelsen Method

Another name for acid-fast staining

Carbol fuchsin- Steam - Acid alcohol- Methylene blue

This is the order of an acid-fast stain.

Carbol fuchsin

Primary dye in acid-fast stain

Steam

Used to help primary dye penetrate the cell wall

Acid alcohol

Decolorizer in acid-fast stain

Methylene blue

Counterstain in acid-fast stain

Acid-fast positive

If you see a cerise (pink/violet) color after acid-fast stain, it is...

Acid-fast negative

If you see a blue color after acid-fast stain, it is...

Mycobacterium

Bacteria we use for an acid-fast stain

Spore staining

Type of structural stain used to detect dormant forms of bacteria

Malachite green- Steam- Water- Safranin

This is the order of spore staining.

Schaeffer-Fulton Method

Another name for spore staining

Bacillus and Clostridium

Gram-positive, spore-forming bacteria

Species

A pure culture contains a single microbial ____________.

On top of agar, embedded in agar, and underneath agar

Where colonies can be found after a pour plate

Only on the surface of the agar

Where colonies can be found after a spread plate

Streak Plate Technique

Method of separating a single species from mixed species population

Zone IIIB

Largest area in 3-Zone streak where you should obtain isolated colonies

General purpose media

Type of media that supports the growth of a variety of microorganisms

Selective media

Type of media that use inhibitors to prevent growth of certain organisms

Differential media

Type of media that use indicators to detect changes that have occurred

Combination media

Type of media that are both selective and differential

Complex media

Type of media where the exact chemical composition is not known

Defined media

Type of media where the chemical composition for each component is known

PEA media

Type of media that is selective only; selects for Gram-positives

PEA

Inhibitor in PEA media

Blood Agar

Type of media that is differential only that differentiates between hemolysis patterns

5% sheep red blood cells

Indicator in Blood Agar media

Beta hemolysis

Complete lysis of red blood cells

Alpha hemolysis

Partial or incomplete lysis of red blood cells

Gamma hemolysis

No lysis of red blood cells

MacConkey Agar

Type of combination media used to identify Gram-negative enterics

Neutral red

Indicator in MacConkey Agar

Crystal violet and bile salts

Inhibitor in MacConkey Agar

Eosin Methylene Blue media

Type of combination media that is used to screen for coliforms

Eosin and methylene blue

The indicator and inhibitor in EMB media

Motile

________ organisms can swim through the soft agar and spread away from the inoculation stab line.

Non-motile

___________ organisms remain confined to the path of inoculation by the soft gel of the medium.

Viable cells

Cells that can replicate and form colonies

A/(A+B)

Formula for dilution factor

30-300

A countable plate has ________ colonies.

Litmus milk turns pink

This is what happens to litmus milk after fermentation.

Litmus milk turns blue

This is what happens to litmus milk after peptone deamination.

Litmus milk loses its color and turns white.

This is what happens to litmus milk after reduction.

Litmus milk will turn clear

This is what happens to litmus milk if bacteria have the caseinase enzyme.

Litmus milk starts to curd

This is what happens to litmus milk if there is enough acid produced after fermentation

Phenol red and Durham tube

Indicator in PR sugar fermentation broth

Phenol Red broth turns yellow

This is what happens to Phenol Red broth if an organism ferments

PR broth turns cerise

This is what happens to PR broth if the organism does not ferment

Kligler's Iron Agar (KIA)

Can test for lactose fermentation, glucose fermentation, and sulfur reduction

1% lactose, 0.1% glucose, 1% peptone

"Ingredients" in KIA test

Phenol red and iron

Indicators in KIA test

The KIA media is all yellow

This is what happens to the KIA media if lactose is fermented

The KIA media is yellow on the bottom, and red at the top

This is what happens to the KIA media if only glucose is fermented

The KIA media starts to undergo reversion causing it to be red

This is what happens to the KIA media when bacteria produce weaker acids during fermentation.

The KIA media turns black

This is what happens to the KIA media during sulfur reduction.

Lipase Plate

Type of differential media used to test for an enzyme that hydrolyzes fats

Spirit blue

Indicator in lipase plate

The plate turns blue.

This is what a positive result on a lipase plate looks like.

Milk Agar Plate

Type of differential media that tests for enzyme caseinase

The plate has clearing around the colony.

This is what a positive result on a milk agar plate looks like.

Starch Agar

Type of differential media used to test for amylase

Iodine

This must be added to starch agar after colony growth as the indicator

Aerobic respiration

Type of respiration that requires oxygen

Anaerobic respiration

Type of respiration that does not require oxygen

Hydrogen peroxide

This is what needs to be added during the catalase test.

Catalase

This enzyme converts hydrogen peroxide into water and oxygen

The plate starts to bubble

This is what a positive result for a catalase test looks like.

Dimethyl-p-phenylenediamene hydrochloride

Indicator in oxidase test

The cotton swab turns dark blue to black

This is what a positive result for an oxidase test looks like

The broth turns red after adding ONLY Nitrate I and II.

This is what one of the positive results in a nitrate test looks like.

The broth turns red after adding Nitrate I, Nitrate II, and zinc.

This is what a negative result in a nitrate test looks like.

Nitrate reductase

Enzyme that reduces nitrate to nitrite; what is being detected in Nitrate Broth