Labs 12-17 Flashcards
What color is Serratia marcescens
Red
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
What color is Micrococcus luteus
Yellow/cream
Properties of agar
Polysaccharide produced by seaweed, adds no nutrient value
Melting point, solidifying point, pour temp of agar
Melting: 100C; solidifies: 42C;
Pour at 45-50C
What is the goal of a streak plate
To obtain a pure culture
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
Why should agar plate be incubated upside down
To prevent condensation
Subculturing technique
Transfer a pure colony from plate to TSA slant
What number of colonies are considered statistically valid
30-300 CFU’s
The SPC method determines what type of cells
Only viable (live)
Optical density (absorbance) determines what type of cells
Both living and dead
What method is most common for determining bacterial numbers
The standard plate or viable count
Explain the relationship between turbidity and % T
As turbidity increases %T decreases
Formula to calculate optical density (OD)
OD = 2 - log (% T)
Ex: % T = 41.4
OD = 2 - log (41.4)
OD = 0.383
Describe the relationship between %T and OD
As %T goes up OD goes down
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.
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
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
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
What was the genus and species of motile organisms used in lab
Proteus mirabilis
Pros and cons of using a wet mount to determine motility
Pro: doesn’t require incubation
Con: dehydrates quickly, spill possibility, Brownian motion
Pros and cons of using soft agar stab to determine motility
Pro: safest
Con: requires 18 hour incubation
What is the full name of the medium we used to determine oxygen requirements
FTM; fluid thioglycollate medium
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
