micrb lab flashcards
How do you aseptically handle a broth culture
Flame loop cool it remove cap flame tube lip take sample flame lip again recap and reflame loop
At what angle should a broth tube be held
About 45 degrees
What should you never do with the tube cap
Do not place it on the bench
Why do you flame the lip of a glass culture tube
To reduce contamination before and after sampling
What note applies to disposable plastic tubes
Do not flame the lip because it will melt
How do you aseptically pick a colony from a plate
Flame loop cool it open lid slightly near flame pick a small portion of colony and close lid
How much colony is usually picked
A very small amount less than the size of a pen tip unless a heavy inoculum is needed
What should always be done with the loop after use
Flame it to sterilize and let it cool before putting it down
What good lab practices help reduce contamination
Wash hands wear PPE tie hair back tidy workspace disinfect bench work quickly and avoid talking over sterile materials
When should you wash your hands in lab
Before beginning and after completing an experiment
Where should long hair be kept in lab
Tied back and if long enough in a bun
Why should you work close to the flame
To stay within the aseptic zone
Why should multiple organisms be handled individually
To prevent cross contamination
What should be done with experimental waste
Discard it in the appropriate receptacles
Where are agar plates labeled
On the bottom agar side not the lid
Why are plates labeled on the bottom
The lid can be switched or removed
What should be included on a plate label
Your name TA or section sample identity date and agar type
What kind of marker should be used on plates
Permanent marker
What is streak plating used for
To dilute bacteria and isolate single colonies
What is the goal of streak plating
Single cells grow into isolated colonies
What is a pure culture
A culture containing only one bacterial species
How many streak sections are used in this lab
Three sections
How is the first streak done
Streak the first third of the plate in a zig zag pattern
Why is the first streak the densest
It deposits the most bacteria for later dilution
What is done between each streak section
Flame the loop and let it cool
How is the second streak done
Cross the first section 1 to 2 times then streak a fresh area without crossing earlier streaks again
How is the third streak done
Cross the second section 1 to 2 times then streak the final area without crossing older streaks again
What indicates successful streak plating
Isolated colonies appear in the later streaks
What if there are too many colonies in the third streak
Use less inoculum cross less from the previous section or make the third zone larger
What if there are too few colonies in the third streak
Use more inoculum and cross more from previous streaks
What is a common reason for little or no growth after streaking
The loop was too hot and killed the cells
How should the loop be held while streaking
Like a pencil with a gentle touch
Why should you avoid pressing hard into the agar
It gouges and damages the agar
How can you organize the streak plate if you run out of room
Draw a T on the plate to define sections
How are plates incubated after streaking
Agar side up in a bag
Why are plates incubated agar side up
To prevent condensation from dripping onto colonies
What are standard plate incubation conditions in this lab
37°C for 24 hours unless otherwise stated
Why are incubated plates bagged
To reduce drying and contamination
What happens to plates after incubation
The TA moves them to 4°C for storage
How should plates be bagged
With the agar side up and not taped together
What are the three lens systems in a compound light microscope
Condenser objective and ocular lenses
What does the condenser lens do
Focuses light onto the specimen
What does the objective lens do
Magnifies the image of the specimen
What does the ocular lens do
Further magnifies the image for viewing
What does the field iris diaphragm do
Adjusts how much light reaches the specimen
What is Köhler illumination
Proper focusing and alignment of light for even illumination and good contrast
Why must the objective lenses be treated carefully
They are the most delicate and expensive part of the microscope
What is the total magnification with a 100X objective and a 10X ocular lens
1000X
What should you look for on a slide when determining cell arrangement
An area where cells are spread out and not clumped
What is cell morphology
The shape and arrangement of individual bacterial cells
What are the two main bacterial cell shapes emphasized here
Coccus and bacillus
What is a coccus
A spherical bacterial cell
What is a bacillus
A rod shaped bacterial cell
What is a diplococcus
Two cocci together
What are tetrads
Groups of four cocci
What is a sarcina arrangement
A packet like cluster of cocci
What are streptococci
Chains of cocci
What are staphylococci
Grape like clusters of cocci
What is a coccobacillus
A very short oval rod
What are diplobacilli
Pairs of bacilli
What are palisades
Bacilli arranged side by side like a fence
What are streptobacilli
Chains of bacilli
What is colony morphology
The visible appearance of a colony on agar
Why is colony morphology useful
It helps describe bacteria and recognize possible pure cultures
What colony texture terms are used
Smooth rough glistening and dull
What does smooth mean in colony morphology
An even regular surface
What does rough mean in colony morphology
An uneven irregular surface
What does glistening mean in colony morphology
A shiny surface
What does dull mean in colony morphology
A surface lacking shine
What colony transparency terms are used
Opaque translucent and iridescent or metallic
What does opaque mean
Light does not pass through
What does translucent mean
Light passes partly through
What does iridescent or metallic mean
The colony reflects light like a colored sheen
What overall colony form terms are used
Circular punctiform spindle filamentous rhizoid and irregular
What elevation terms are used
Flat raised convex pulvinate and umbonate
What margin terms are used
Entire undulate lobate filamentous erose and curled
What does entire margin mean
A smooth even edge
What does undulate margin mean
A wavy edge
What does lobate margin mean
An edge with rounded lobes
What does filamentous margin mean
A thread like edge
What does erose margin mean
A serrated or jagged edge
What does curled margin mean
An edge with concentric ring like waves
What is the difference between cell morphology and colony morphology
Cell morphology describes individual cells under the microscope while colony morphology describes visible colony appearance on agar
What is a simple stain
A stain where all cells on the slide stain the same color
What are common simple stains
Methylene blue crystal violet and safranin
What is a positive stain
The cells are stained while the background remains clear
What is a negative stain
The background is stained while the cells repel the stain
What is a differential stain
A stain that distinguishes different cell types or structures by staining them differently
What is the most important differential stain in this course
The Gram stain
What does the Gram stain tell you
Whether bacteria are Gram positive or Gram negative based on cell wall structure
What color are Gram positive bacteria after a Gram stain
Purple
What color are Gram negative bacteria after a Gram stain
Pink
Why do Gram positive bacteria stay purple
Their thick peptidoglycan traps the crystal violet iodine complex
Why do Gram negative bacteria turn pink
Alcohol removes the primary stain and they take up the safranin counterstain
What are the Gram stain reagents in order
Crystal violet iodine ethanol and safranin
What does crystal violet do
Primary stain colors all cells purple
What does iodine do
Acts as a mordant and fixes crystal violet in the cells
What does ethanol do in Gram staining
Decolorizes Gram negative cells by removing the crystal violet iodine complex
What does safranin do
Counterstains decolorized cells pink
Why is decolorization timing critical
Too long can remove stain from Gram positive cells and too short can leave Gram negative cells purple