MCB Practical

Turbidity 

The haziness or cloudiness of a liquid. The more turbid the sample is, the more bacteria is present

Lag Phase

Cells are adjusting to new conditions and synthesizing metabolic enzymes

Log Phase

Period of rapid division and exponential growth

Stationary Phase

Cell division slows due to limited nutrients and/or build up of waste. New cell growth is matched by an equal amount of cell death.

Death Phase

cells are dying due to limited nutrient and/or buildup of waste products

Generation Time

The amounf of time it takes for one cell to divide into two

Solid Media (Agar plates)

used to isolate bacteria

Liquid (broth) Culutre media

used to grow cells to high densities

T-Streak Plate Method

The quickest and most economical method of bacterial isolation

Simple Stain

Staining procedres which only use one stain

Direct Stain

A simple stain that colors the bacteria

Negative Stain

A simple stain which stains the background but leaves the bacteria unstained

Liquid Media

Liquid media such as nutrient broths are used to grow large numbers of orgnaisms and are used for various experiments and diagnostic tests

Media

Microorganisms are commonly grown on a variety of different food scources called media

Solid Media

Used to examine colony morphology of organisms and to help in isolating organisms. Most common solidifying agent added to media is agar. 

Complex

Non-synthetic or undefined; their exact chemical composition is not known

Synthetic

Defined media; made up of a strict list of inorganic compounds and carbon sources

General Purpose Media

Used to grow up dense cultures of bacteria, support a wide range of organisms. Some examples are nutrient borht, nutrient agar, and tryptic soy broth.

Selective Media

they select for a certain type of microbe by supplying optimal conditions for its growth, while selecting against some potential competitors form the same habitat.

Differential Media

They allow certain bacteria to be differentiated from other, typically by a change of color, production of a cone clearing, etc.

What was the BPB Broth used to select for?

Enterococcus faecalis

MacConkey Agar

Example of differential media. Organisms that femrment lactose produce pink colonies and ones that don’t ferment lactose produce non-pink colonies.

Fermentation

Inneficient process involving anaerobic oxidation of substrates (sugars)

Products of fermentation

Always acid and sometimes gas

What does motility allow bacteria to do?

To colonize new environments, find nutrients, and escape harmful compounds

What is the most common organelle of bacterial motility?

Flagellum

Flagellum

One or more long protein filaments which rotate, causing cell motion.

Motility

The movement of bacteria under their own power, it is not random movement. 

BPB Broth Results

• Purple color : negative (no growth of bacteria)

• Yellow color: positive (growth of bacteria)

Citrate Agar Test Results

• Green: negative (no growth)

• Blue: positive (growth, alkaline)

• Yellow: positive (growth, acidic)

Exoenzyme

Enzymes that are secreted by the cell into their environment

Hydrolysis

Break down

Amylase

catalyzes starch hydrolysis

Casease

a collection of enzymes that hydrolyze milk protein, casein, into peptides and ultimately amino acids

Gelatinase

An enzyme that hydrolyzes gelatin

Catalase

An enzyme which protects cells from hydrogen peroxide

Oxidase

An enzyme which indicated the presence of cytochrome c, a component of some electron transport systems

Starch Plates Results

• If there is a red halo around the bacteria that means that it is an amylase positive bacteria

• If there is no red halo around the bacteria that means that is amylase negative

Casein Plates Results

If the casein has be hydrolyzed then there will be a clearing around the growth, this is because the caseinase enzyme was present

Gelatin Tubes Result

• Liquid media: positive, gelatinase is present

• Solid Media: Negatie, gelatinase is not present

Catalase Test Results

If there is immediate bubble formation then it is catalse positive, if there is no bubble formation then it is catalase negative

Oxidase Test Results

If there is a blue color change within 30 seconds then it would be oxidase positive and cytochrome c is present. No color change means oxidase negative and no presence of cytochrome c

Fermentation Test Results

• If the tube is any color other than red that means that it is positive for growth

• If the tube color is yellow, salmon, or orange that means that acid was a product, if the color is red or magenta then acid is not a product

• If there is a gas bubble then is it positive gas production

Motility Test Results 

• A: Control

• B: Positive; not strict aerobe

• C: Negative

• D: Positive; obligate aerobe, moves to the top of the tube

Nutrient Agar Plate Results

It is just positve or negative for growth, if there is growth then its positive, if there is no growth then it is negative

MacConkey Agar Test Results

• If there is growth on the plate then it would be positive for growth if there is no growth on the plate then it would be negative for growth

• Color Change: if the color is pink then it is more acidic and it ferments lactose, if the color is not pink then it is neutral and does nto ferment lactose

Gram Positive Bacterial Cells

They have a thick peptidoglycan layer and retain crystal violet, so they are purple

Gram Negative Bacterial Cells

They have a thin peptidoglycan layer and an outer membrane. The crystal violet washes out and safranin counterstains the membrane, they are pink

Gram Stain Procedure

• Add 1-2 drops of crystal violet and wait 1 minute and then rinse the off with water

• Add 1-2 drops of grams iodine, wait 1 minute and then rinse off with water

• Add 1-2 drops of ethanol and immediately wash off with water

• Add 1-2 drops of safranin wait 45 seconds and then rinse off with water

• Dry the slide 

Spore Stain

• Identifies endospore-forming bacteria

• certain bacteria sporulate under environmental stress

  • limited carbon sources (such as nutrient agar plates w/o glucose)

• bacteria have evolved to sporulate as a mechanism to preserve their DNA when under environmental stress

• Heat used during staining does not cause sporulation

Spore Stain Procedure

• Prepare a slide, use bacterial smears not liquid media and let them air dry

• Place a peace of pre-cur bibulous paper over the smear but do not heat fix

• Add about 3-5 drops of 5% aqueous malachite green dye over the paper and let it sit for 5 minuutes

• Using the slide holder, place the slide onto the aluminum tray on the hot plate

• Monitor the bibulous paper and keep adding the dye, as needed, to keep the paper from drying, continue steaming for 3-5 minutes

• After steaming discard the paper into the malachite green waste beaker and let the slide cool

• Counterstain the smear with safraning and let it sit for 2 minutes, no heating. 

• Rinse the safranin and dry. 

Spore Stain Results

• If the bacterial species forms endospores, there should be green enedospore inside (and sometimes outside of) pink vegetative cells.

Coliform

Facultativelty anaerobic, gram-negative, noHEterendospore forming, rod-sahped bacteria that ferment lactose with acid and gas formation

Heterotrophic Plate Count

Quantitative method used to determine the overall quality of the water as well as the effectiveness of disinfeectants used in water treatments

Presumptive Test

Uses lauryl-tryptose (LT) broth which is

• selective for intestinal bacteria (sodium lauryl sulfate)

• differential gas production during lactose fermentation (durham tube) 

• Looking for gas bubbles created by lactose fermentation, which indicate possible coliform growth