MICROBIO LAB MIDTERM EXAM

Laboratory Techniques

To study microorganisms, it is helpful to be able to grow them. What kind of medium are microorganisms grown on?

Culture medium

type of medium which contains nutrients, such as carbon and nitrogen, required for the cells to grow

culture medium

type of medium composed of digests of chemically undefined substances, such as yeast and meat extracts or digests

complex medium

type of medium for which a precise chemical composition is known

defined medium

examples of complex, general-purpose media that serves for the isolation and cultivation of a variety of microorganisms

Nutrient Agar + Trypticase Soy Agar

a complex medium used when cultivating fungi or yeast

Sabouraud Dextrose Agar

A liquid in which a culture medium can be prepared in

broth

A medium that contains a solidifying agent (usually agar) in which a culture medium can be prepared in

solid medium

Agar is extracted from what organism?

Rhodophyta (italicized)--red algae

Why is agar a good solidifying agent?

After being heated to 100 degrees celsius, agar will solidify at 40-42 degrees celsius and will not melt again until the temperature reaches 80-90 degrees celsius.

solid media in petri dishes

plates

What are the surface of plates used for?

Used for the isolation and growth of microorganisms

Test tubes containing solid medium, which has been allowed to solidify at an UPRIGHT position

deeps

What are deeps usually used for?

anaerobic growth

test tubes containing solid medium that has been allowed to solidify ar at an ANGLE

slants

What are slants used for?

Used for the maintenance of stock cultures of microorganisms

Media can be…?

selective, differential, both, or neither

a nutrient medium designed to favor the growth of certain microbes and to inhibit undesirable competitors

selective medium

a medium which provides a visible indication of a physiological characteristic of a microorganism

differential medium

Could be demonstrated on differential media

carbohydrate fermentation or enzyme production

this agar is selective ONLY and selects for gram-positive organisms, but tells nothing of the physiology of the organism growing on it

Phenyl ethyl alcohol agar (PEA)

this agar is BOTh selective and differential and selects for the growth of salt-tolerant organism and differentiates mannitol fermenters from non-mannitol fermenters

Eosin methylene blue agar (EMB)

turns agar from red to yellow

mannitol fermenters

does not change color of agar

non-mannitol fermenters

is BOTH selective and differential and selects for Gram-negative bacteria and differentiates lactose fermenters from non-lactose fermenters

Eosin methylene blue agar (EMB)

Why is it necessary to sterilize media and any object with which will come into contact with it?

to protect against contamination by unwanted organisms

the killing or removal of all living organisms and their viruses from a growth medium

Sterilization

a method of sterilization that uses a sealed device to allow for the entrance of steam under pressure

autoclave

What is the autoclave heated to? For how long?

121 degrees celsius at 15 pounds per square inch (psi) pressure for 15 minutes-1 hour (depending on what is being sterilized and how much)

__ media takes longer to reach 121 degrees celsius than __ media

solid; liquid

__ volumes take longer to reach 121 degrees celsius than __ volumes

larger; smaller

a method that is frequently employed to sterilize liquid media and other items that are heat-resistant

autoclave

type of sterilization that kills by oxidation effects

dry heat sterilization

one of the simplest methods of dry heating sterilization

direct flaming

procedure used many times this semester when inoculating loops and inoculating needles by holding it in the flame of a Bunsen Burner until it is glowing red hot

direct flaming

Why is it important to flame the entire wire loop or needle?

to minimize contamination

used to sterilize items like glassware by placing them in an oven and heating it to 170 degrees celsius for 2 hours

hot-air sterilization

process used to sterilize heat-sensitive liquids or gases

filtration

a device with pores too small for the passage of microorganisms but large enough to allow passage of the liquid or gas

filter

a tough disk composed of cellulose acetate or cellulose which contains a large number of tiny holes

membrane filter

What does the surface of a filter do?

traps particles while allowing the liquid to pass through; a physical; removal of bacteria or other contaminants

Example of filter sterilization:

beer

used to sterilize other heat sensitive objects such as plastic petri dishes

gas chemo-sterilizers

one of the widely used gas

ethylene oxide

made the use of plastic petri dish and plastic syringe possible

ethylene oxide

type of gas that is toxic and explosive which is released into a tightly sealed chamber where it circulates for up to 4 hours with the items to be sterilized

ethylene oxide

used to sterilize paper, leather, wood, metal, and rubber products

ethylene oxide

is used by NASA to sterilize interplanetary spacecraft

ethylene oxide

type of sterilization process in which no heat is used

cold-sterilization

used in sterilizing many substances and includes microwaves, ultraviolet radiation, X-rays, gamma rays, and electrons

Electromagnetic radiation

causes damage to the DNA (thymine dimers) leading to the death of exposed organisms

Ultraviolet (UV) radiation

cannot penetrate solid, opaque, light-absorbing surfaces and is useful only to sterilize exposed surfaces

Ultraviolet (UV) raduiation

causes ions and other reactive molecules to be produced

ionizing radiation

can degrade or alter biopolymers such as DNA and proteins

Ionizing radiation

can be used in the food industry and for sterilization and decontamination of medical supplies

Radiation

Microorganisms are ____ in the environment

ubiquitous -- (meaning found everywhere)

Unwanted organisms that contaminate microbial cultures or sterile surfaces

contaminants

How do we avoid contamination by contaminants?

by utilizing proper aseptic techniques while transferring cultures or inoculating sterile materials

refers to a procedure performed under sterile conditions

aseptic technique

Steps to help prepare the work area for aseptic technique:

By cleaning the bench area and outfitting yourself appropriately:


1. Upon entering the lab, the lab bench should be wiped with disinfectant and a clean paper towel to eliminate contaminants on the surface
2. Keep on the lab bench ONLY those items needed to perform the experiment
3. Tie back long hair to keep it out of the work area
4. Gloves should be worn during experiments

What type of sterilization does aseptic technique for using culture require?

dry heat sterilization

When must the inoculating loop be sterilized?

must be sterilized before being used to transfer bacterial cultures and after culture transfer

process commonly used to sterilize a loop

flaming

How do you flame a glass culture tube?

pass the neck of the culture tube briefly through a flame both before and after a loop or pipette is placed into the tube

How do you flame a plastic culture tube?

you don’t. plastic should not be passed through the flame. Passing plastic will cause the plastic to melt if passed through the flame too slowly

How should tubes or flasks containing bacterial culture be held? Why?

should always be held at an angle to prevent contaminants from the air from entering the tube

What do you do with the cap of the tube while flaming?

hold it. It should never be placed on the bench top, but held in the same hand as the inoculating loop

When should we utilize proper aseptic techniques in the lab? Why?

at all times; to prevent contamination of the stock cultures and the media you are inoculating

Steps for aseptic technique in transferring culture from a tube to other media or a microscope slide:

1. gently shake the culture tube from side to side to suspend the microorganisms. Do not allow liquid to rise to the cap of the tube
2. heat the loop and wire to red-hot. Flame the handle slightly also
3. Remove the cap and flame the neck of the tube. Do not place the cap down on the table
4. after allowing the loop to cool for at least 5 seconds, remove a loopful of microorganisms. Avoid touching the sides of the tube.
5. Flame the mouth of the culture tube again
6. return the cap to the tube and place the tube in a test-tube rack
7. place the loopful of microorganisms in the center of the target circle on the slide for a smear, onto an agar plate, or into a tube of broth, slant, or deep
8. Flame the loop again before removing another loopful from the culture or setting the inoculating loop aside

How should you properly handle a petri dish while inoculating?

1. Keep the lid on the plate as much as possible. Never completely remove the lid
2. always check the loop for coolness by touching the medium in an uninoculated area before streaking

Why do we view bacterial cells under magnification?

to observe their relative size, shape, and arrangement

Why must adequate magnification be used when viewing prokaryotic cells

because of their small size (most are 0.2 micrometers to 10 micrometers)

must be used to observe microorganisms and the total magnification achieved is 1000x

oil-immersion objective

Why is it that, even under adequate magnification, unstained cells may be difficult to see?

because of lack of contrast and because cells consist of mostly water

process done to make cells more visible

staining

What is the first step to staining cells?

making a smear

can be made from cultures in liquid or on solid medium

smear

procedure varies slightly with the culture medium used and with the direct or indirect stain procedure

smear

what is the process for a direct stain?

1. slide is labeled with the culture to be stained
2. the smear is made
3. smear is dried
4. smear is heat fixed using the slide warmer

process which kills the cells, destroying autolytic enzymes, and helps the cells to adhere to the slide

heat fixation

What makes cells naturally sticky?

proteins and sugars on their cell surface

Why is adhesion of the cells to the slide essential?

necessary or the cells will be washed from the slide during staining

What is the process for indirect stains?

1. smear is made in the primary stain
2. smear is never rinsed in the staining procedure
3. heat fixation is not required

Difference between indirect and direct staining?

Direct staining requires heat fixation

Material needed to prepare a smear

1. microscope slides
2. inoculating loop
3. bunsen burner
4. marking pencil
5. clothespin
6. microbial cultures

Process of creating a smear from LIQUID media:

1. two loopfuls of liquid are placed in the center of the “target circle”
2. organisms are dispersed over the entire area of the “target circle”
3. (evaporation of water)
4. the smear is allowed to dry on the slide warmer

Process of creating a smear from SOLID media:

1. Two loopfuls of water are placed in the center of the “target circle”
2. a very small amount of orgnaism is dispersed with the inoculating loop in water over the entire area of the “target circle”
3. (evaporation of water)
4. the smear is allowed to dry on the slide warmer

In making a smear, what should we consider?

culture concentration

A colony on a plate can have a million of cells depending on….?

colony size and the size of cells forming the colony

What happens if you use too many cells from a colony, slant, or broth culture in your smear preparation?

you will end up with a thick smear that is difficult to decolorize in differential staining

Where is a thick smear best viewed?

on the edges of the smear where there are fewer cells and the staining procedure most reliably done

What has fewer cells? A loopful from a turbid broth or a colony on a plate?

loopful from turbid broth

Why may cells be difficult to see even under adequate magnification?

because cells are 90% water by weight

makes cells more visible by providing contrast

staining

What factors can be used to differentiate bacteria?

morphology, shape, chemical composition, biochemical activities, and other factors

Staining allows the ____ of a cell to be determined

morphology

What are the basic shapes of most bacteria that we will encounter in this course?

coccus (pl. cocci), rod/bacillus (pl, bacilli), and spiral

cells arranged as pairs

diplo

cells arranged in chains

strepto

cells arranged in clusters

staphylo