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.