Microbiology Lab Exam #1
true or false - An inoculating loop is sterilized in a flame by briefly passing it through the flame three times.
false
true or false - It is OK for one group member to hold a test tube while another member extracts bacteria from it for a transfer.
false (will accept true)
some of the most wildly consumed
fermented foods
the three common bacterial shapes
coccus bacillus spiral
colonies of bacteria are referred to _.
colony forming units (CFUs)
color of gram neg. cells after decolorizing
colorless
Plate counts tend to underestimate microbes. (In fact, studies show only 1-10% of soil organisms have ben isolated and grown in culture) Why?
Some microbes have very specific growth requirements.
three major areas of concern when referring to food microbiology
food production food spoilage food-borne illness
fx of ocular
furthers magnifies image (10x)
bacteria that retain the safranin stain, making them appear pinkish red
gram negative
fx of condenser
concentrates light to focus on specimen
Why does the capsule stain method require heating during the primary staining step? What fx did the bibulous paper serve?
Heat allows the stain to stick, and the bibulous paper keeps stain there and moist while steaming.
magnification of high objective lens
40x
Why is the gram stain so important?
-It's one of the most commonly used differential bacterial stains. -It is often the first step in bacterial identification.
What are some benefits bacteria gain from having a capsule?
-allow bacteria to escape from phagocytic cells -allow bacteria to resist desiccation -aids bacteria in attachment
Describe the steps of putting away the microscope correctly.
-remove slide -clean all objective lenses with lens paper and lens cleaner -put lowest power objective over stage -lower stage -turn off light -wrap the cord around the base -cover microscope with dust cover
ocular lens magnification
10x
simplistic single-celled organisms
bacteria
fx of objective
creates a magnified image and refracts light
stain that is anionic
negative stain
non-acid fast bacteria
Staphylococcus aureus
Why do the capsules show up with the capsule stain technique when both the background and the cells themselves are stained?
The capsules can be observed using a modified negative stain. Detecting a capsule involved staining both the background and the cell while the capsule itself remains unstained.
What is the difference between preparing a smear from a solid media and preparing one from a liquid media?
The liquid media does not need a water droplet.
Why do gram positive cells stay purple?
The teichoic acids of the gram pos. cell wall will react with the crystal-like complexes formed by the mordant (iodine) and help them be retained in the cell. Also, the thick layers of PTG are resistant to the ethyl alcohol.
Why are endospores important to bacterial cells?
They are dormant structures that provide protection for cells exposed to harsh environmental conditions.
Why are endospores not considered to be true reproductive spores?
They have a vegetative cell that grows, metabolizes, produces one spore, then dies.
Why do gram neg. cells turn red when stained?
They only retain the safranin counterstain.
Why is microbial fermentation beneficial to certain food groups?
They provide characteristic flavors, aromas, and textures of certain foods like yogurt and bread.
What disinfectant do we use and when should you use it during a bacterial transfer activity?
We use 10% bleach before beginning after finishing the activity and in case of spills.
Why is it important to employ aseptic techniques when transferring bacteria?
We want to make sure we only have the bacteria present that we're testing. Without aseptic techniques, multiple bacteria could make its way into the sample being tested.
Why should you wait for your loop to completely cool before attempting a bacterial transfer?
You could kill the bacteria.
How do you prevent air contamination of agar plates if they are not flamed?
You partially open the lid so that little to no contamination affects the agar plate experiment and work near bunsen burner in sterile air.
chemical used to decolorize acid fast stain
acid alcohol
What do acid fast and non-acid fast bacteria look like?
acid fast: red non-acid fast: blue
Microbes play important roles in __.
agriculture nutrient recycling sewage processing commercial food production industry
List some examples of diseases caused by bacteria that produce spores.
anthrax tetanus botulism
antimicrobial chemicals considered safe for use on living tissues
antiseptics
any technique employed to avoid contamination
aseptic technique
All growth media should be sterilized to ensure a __.
axenic culture
pure culture of organism of interest
axenic culture
measure of the variety of organisms
biodiversity
color of non-acid fast stain rxn
blue
What is the primary stain for the acid fast stain? what is the counterstain?
carbolfuschin; methylene blue
factors that affect biodiversity in soil
climate texture vegetation chemical conditions (pH, nutrient availability, salinity) physical conditions (water availability and temp)
stain for capsule stain
crystal violet
What is the primary stain for the gram stain? What is the counterstain?
crystal violet; safranin
Structural stains that detect specific physical or chemical characteristics of a cell are sometimes referred to as __.
differential stains
What is a pair of rod-shaped cells called?
diplobacilli
What is a pair of round bacterial cells called?
diplococci
antimicrobial chemicals meant for use on inanimate surfaces
disinfectants
What test do we use to assess the effectiveness of a specific antiseptic or disinfectant at inhibiting microbial growth?
disk diffusion test
chemical used to decolorize gram stain
ethyl alcohol
True or False? Bacterial endospores are true reproductive spores.
false
true or false - When you remove the lid of a test tube or agar plate, it is OK to set it on a clean work bench wile you remove or add bacteria.
false
bacteria that retain the crystal violet stain, making them appear purple
gram positive
Name some environmental pressures that Myobacterium and Norcardia bacteria can resist thanks to the thick mycolic acid layer in their cell walls.
heat cold desiccation disinfectants and antibiotics
fx of stage
holds specimen
What is the mordant for the gram stain and what is its fx?
iodine; to form crystal-like complexes that stay because they are not easy to remove from the cell
What do we hope to accomplish by heat-fixing a bacterial sample?
kill bacteria cells and adhere them to the microscope slide
stain used for the spore stain
malachite green
Can the coarse focus knob be used when adjusting the focus of any objective lens?
no
total magnification is determined by multiplifying the magnification of the __ lens by the magnification of the __ lens.
ocular; objective
means the image should stay in focus when you changes lenses
parfocal
how to properly sterilize an inoculating loop
pass the inoculating loop through the flame of the bunsen burner and allow the loop and upper part of the wire to get red hot
common method for enumerating bacteria in soil and food samples
plate count
Does gram positive or negative have more PTG in its cell wall?
positive
Is Staphylococus aureus gram positive or negative?
positive
color of gram pos. stain rxn
purple
fx of coarse adjustment
raises and lowers stage
color of acid fast stain rxn
red
color of gram neg stain rxn
red
fx of diaphragm
regulates amount of light through condenser
Which stain do the vegetative cells retain?
safranin
performed in order to isolate 30-300 CFUs on petri plates
serial dilutions
fx of fine adjustment
sharpens the image
stain that is basic (cationic)
simple stain
What are the two stain techniques we will utilized during this class (not gram stain)?
simple stain negative stain
What is a chain of round bacteria called?
streptococci
stain that detects specific physical or chemical characteristics of a cell that may be useful in removing some of the ambiguity of bacterial identification
structural stain, differential stain
What compound is found in the gram positive cell wall but not the gram negative cell wall and reacts with the crystal violet-iodine complex and helps it be retained by the gram positive cell?
teichoic acids
simple vs negative stains: which dye is attracted to the negative nature of the cell? why?
the basic dye (cationic) -> it's postively charged, so it's attracted to the negative
simple vs negative stains: which dye binds to the slide and why?
the basic dye (cationic) binds to the cell so that it takes on the color of the dye and becomes visible using microscopy
What makes acid fast bacteria like Mycobacterium so resistant to staining?
the thick mycolic acid layer in the cell wall
True or false - The total magnification a microscope is capable of is only limited by the magnifying power of the objective and ocular lenses.
true
True or false - When you observe a specimen at a higher power, the field of view is actually smaller even though the specimen appears larger.
true
true or false - The opening of test tubes and the lid should be passed through the flame before the lid is replaced on the tube.
true
means "found everywhere"
ubiquitous
types of food where microbial fermentation would be used to provide characteristic flavors, aromas, and textures
yogurt and bread
What is the name of the area around the filter disk with no bacteria growth called?
zone of inhibition
6 steps to follow aseptic technique
1) sterilize work area 2) light bunsen burner and sterilize inoculating loop (wait for loop to cool) 3) grab your tube containing the bacteria sample, take top off, and sterilize rim 4) insert loop (grabbing tiny amount of bacteria), remove loop, sterilize rim of tube, and replace top 5) grab petri dish, tilt lid up like clam, carefully streak agar, replace lid, and invert petri dish 6) sterilize inoculating loop and work area, once again
magnification of oil immersion
100x
magnification of low objective lens
10x
Where should you label your agar plates and how should they be stored?
Agar plates should be labeled on the side of the agar on/around the rim of the container and stored upside down.
Why is a gram stain often the first step in identification of bacteria in a clinical sample?
Certain antibiotics can be prescribed since particular antibiotics work more effectively on gram + and gram - cells.
For the soil part of the lab, why did we use three different media even though we all started off with 1g of the same soil?
Different media promote growth of different microbes.
What does it mean to say a cell is gram variable and what are the two possible causes?
Gram positive stains that don't have consistent amounts of teichoic acids can be seen as gram negative reaction. -- could be due to genetics or could be due to the age of the culture
How does oil immersion improve resolution?
It improves resolution and reduces light refraction.
acid fast bacteria
Mycobacterium smegmatis
Why should you be careful not to overheat the smear during a capsule stain?
Overheating the smear can cause the capsule to shrink.