Labs 12-17 Flashcards by Apryl Lytle

What color is Serratia marcescens

Red

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What is a pure colony

Colony is assumed to be the identical progeny of the original cell and can be picked and used for further study

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What color is Micrococcus luteus

Yellow/cream

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Properties of agar

Polysaccharide produced by seaweed, adds no nutrient value

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Melting point, solidifying point, pour temp of agar

Melting: 100C; solidifies: 42C;

Pour at 45-50C

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What is the goal of a streak plate

To obtain a pure culture

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What assumptions are made about each colony

Colony is assumed to be the identical progeny of the original cell and can be picked and used for further study

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Why should agar plate be incubated upside down

To prevent condensation

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Subculturing technique

Transfer a pure colony from plate to TSA slant

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What number of colonies are considered statistically valid

30-300 CFU’s

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The SPC method determines what type of cells

Only viable (live)

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Optical density (absorbance) determines what type of cells

Both living and dead

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What method is most common for determining bacterial numbers

The standard plate or viable count

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Explain the relationship between turbidity and % T

As turbidity increases %T decreases

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Formula to calculate optical density (OD)

OD = 2 - log (% T)
Ex: % T = 41.4
OD = 2 - log (41.4)
OD = 0.383

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Describe the relationship between %T and OD

As %T goes up OD goes down

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Describe the procedure for using the spec 20

Adjust the left knob to zero, insert blank and adjust the right knob to 100. Insert sample, close cover and read the %T.

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Factors to keep in mind when using a wet mount to view motility

Examine under an oil immersion objective; use darkfield optics to view; look for directional movement; always examine immediately because motility decreases with time

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Several facts about using hanging drop slides to view motility

Focus near the edge of the drop because most bacteria are drawn to the edge by surface tension; examine it quickly b/c water condensation may develop and decrease clarity

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What is Brownian motion

Movement due to molecular bombardment of cells causing cells to shake or jiggle about but not move in any vectorial way

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What was the genus and species of motile organisms used in lab

Proteus mirabilis

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Pros and cons of using a wet mount to determine motility

Pro: doesn’t require incubation
Con: dehydrates quickly, spill possibility, Brownian motion

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Pros and cons of using soft agar stab to determine motility

Pro: safest
Con: requires 18 hour incubation

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What is the full name of the medium we used to determine oxygen requirements

FTM; fluid thioglycollate medium

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What is the ingredient in FTM that acts to lower the O/R potential (oxygen amounts)

Sodium Thioglycollate

What is the meaning of the pink (red) color at the top of the tube of the medium FTM

An indicator for the presence of oxygen

Name of the indicator dye in FTM and what does it detect

Resazurin which is an indicator for the presence of oxygen

Why is FTM boiled before inoculation

To eliminate any oxygen present

What are two enzymes lacking in and obligate anaerobe

Catalase and superoxide dismutase

Where would you find obligate aerobic bacteria in the FTM? What environment does it prefer?

Towards the top of the FTM not in the middle or at the bottom. Must grow in oxygen

Where would you find the microaerophiles in the FTM? What conditions do microaerophiles prefer?

A thin line In the middle of the tube. Not the top not at the bottom; prefers oxygen conc of 2 to 10% instead of the 20% found in the atmosphere

Where would facultative anaerobes grow in FTM? What do they prefer?

They would grow throughout the tube. Metabolism is flexible so they can grow with or without oxygen present

Where would anaerobic bacteria grow in FTM? What environment do they prefer?

Only at the bottom of the tube. Cannot tolerate oxygen

Examples of obligate aerobes

Pseudomonas, Micrococcus, and many Bacillus

Examples of a microaerophile

Helicobactor pylori

Example of a facultative anaerobe

Escherichia coli

Examples of aerotolerant anaerobes (also called obligate fermenters)

Enterococcus faecalis, Streptococcus pyogenes

Examples of obligate anaerobes

Clostridium methanococcus and bacteroides

Describe general-purpose media

Designed to grow a broad spectrum of microorganisms. Ex: TSA, TSB

Describe enriched media

Contains an ingredient that will promote a robust growth of bacteria Ex: blood agar

Describe selective media

Formulated to prevent the growth of certain bacteria but not others. Ex: salt agar, manitol salt agar, macConkey agar

Describe differential media

Designed to display visible differences among microorganisms | Ex: MSA, MAC

The selective agents in SA, MSA, MAC

``` SA= 10% salt MSA= 7.5% salt MAC= bile salts ```

What are the differential agents in BA, MSA, MAC

``` BA= blood MSA= sugar (mannitol) and phenol red (pH indicator) MAC= lactose and neutral red (pH indicator) ```

Describe beta hemolysis

Complete hemolysis giving a clear zone with a clean edge around the colony

Describe alpha hemolysis

Incomplete hemolysis producing a cloudy zone of greening around the colony due to the production of methemoglobin

Describe gamma hemolysis

No hemolysis or no change in the blood around the colony

PH indicator in MSA turns what color at what pH

Phenol red turns yellow around 6.8; means fermentation happened

PH indicator in MAC turns what color at what pH

Neutral red changes from yellow to red around 6.8; means fermentation happened

Which organisms do all three selective media select for

Salt (cocci)

What organisms do all three selective media select against

Rods

Which organisms were able to ferment mannitol

Staphylococcus aureus

Which organisms were able to ferment lactose

E. coli

What is the role of CaCl2

Neutralizes the cell membrane

What is special about the MM 294 strain of E. coli

It is not resistant to ampicillin

What is it meant to be competent

Ability to take up free DNA

What is the source of DNA for transformation

P green plasmid

What are the names of the two genes that are found on the P green plasmid

Gene for green fluorescent protein, gene for ampicillin resistance

What proteins do the genes code for

Green fluorescent proteins and ampicillin resistance

What is the purpose of the heat shock step during bacterial transformation

To open the Porins to allow the gene to enter

What enzyme provides resistance to ampicillin

Beta lactamase

What is the mode of action of the enzyme beta-lactamase

Breaks the beta lactam ring

Describe The only plate with no growth

LBA/AMP agar; no p green; is the negative control