microbiology lab exam 1

what are the different types of PPE found in the lab?

goggles, lab coats, close-toed shoes

• clothes that are not too flowy or dangling

• BSL 2 = biosafety cabinets

• BSL 3 = respirators and double gloves

• BSL 4 = positive pressure suits and HAZMAT suits

what are the different biosafety levels? which one is the most dangerous?

BSL 1 = no gloves or goggles, least risk/harm, do not typically cause disease

BSL 2 = may cause disease (salmonella)

BSL 3 = causes potentially lethal disease (COVID)
BSL 4 = causes deadly disease, no treatment or vaccine available

• BSL 4 is MOST dangerous

what are some hazards that you can find in the lab?

sharp objects: scalpels, needles, knives

chemicals: acids, bases, alcohols, preservatives

fire

lab organisms: biohazard, bacteria, tissues, culture cells, dead rats and mice

what are some items not allowed in the lab?

food, drinks, open-toed shoes

what are the steps to wash your hands?

1. remove rings and watches

2. turn on the hot and cold water to get warm water

3. wet your hands

4. apply soap

5. clean under nails with brush or stick

6. lather and scrub hands for 20 seconds at least

7. rinse hands

8. dry hands with paper towel

9. use paper towel to turn off water

10. throw paper towel

what is media?

media is any substance used to grow microorganisms in the lab

• contains nutrients that allow microbes to grow

what is broth versus solid media? what ingredient makes media solid?

liquid media is called broth, solid media is agar

• agar powder can be added to liquid media to make it solid

what is autoclave sterilization? what is ethylene oxide sterilization?

autoclave sterilization = high heat with pressure and steam

• prevents boiling and prevents dry out

• good for media, liquids, plastics

• wet, high heat

ethylene oxide sterilization = gas that kills microbes

• good for solids (clothes)

• can be used for all types of materials: glass, plastics, solids

• BAD for liquids, the gas cannot penetrate liquid

• good for bulk sterilization

what is filter sterilization? when would you use it?

pass liquid through a filter with extremely small pores (0.22 um)

• pores smaller than bacteria

• bacteria are removed from liquid

• good for liquids that contain heat sensitive materials like antibiotics (cannot be autoclaved)

what does sterility mean?

sterilization removes all life forms including spores and viruses

• nothing viable is left behind

what is a contamination?

growth of unwanted microbes

how do you inoculate a broth? slant? deep?

use an inoculating loop to transfer bacteria into broths, slants, and plates

use an inoculating needle to transfer bacteria into deeps

inoculate by:

1. heat sterilize the tool

2. use pinky method to open stock culture tube

3. pass tube over fire

4. stick loop/needle in culture to get sample

5. pass tube over fire and close it, rerack it

6. transfer the sample to the plate or broth

how do you perform a four-quadrant streak?

1. flame loop, get bacteria from a source and introduce to petri dish on one section

2. flame loop, rotate plate 90 degrees, and use loop to spread bacteria from the previous section to the new area (second)

3. flame loop, rotate plate 90 degrees, and use loop to spread bacteria from the second area to the third area

4. flame loop, rotate plate 90 degrees, and use loop spread bacteria from the third area to the fourth and final quadrant

what is a bacterial colony? why is it important to get colonies when streaking for isolation?

a bacterial colony originates from one bacterial cells

• all the bacteria in a colony are theoretically identical to each other

it’s important to get colonies when streaking for isolation because this means we have isolated one bacterial species

when would you use a loop versus a needle?

use an inoculating loop to transfer bacteria to a broth or solid plate/slant

use an inoculating needle to transfer bacteria into a deep

how do you label and store Petri dishes?

label the BOTTOM of the Petri dish with the name of the culture, ensuring the label stays with the culture itself

store Petri dishes upside down so that the condensation gathers on the lid and not on the agar (condensation would contaminate the agar)

what is a pure versus mixed colony?

pure colonies are cultures with only one type of bacteria

• this is the goal so that we know what bacteria we are working with

mixed cultures are mixtures of different types of bacteria

• throat swabs would have a mestizo of bacteria

what are the main parts of a microscope?

• ocular lens: lens you look through

• objective lens: lens that faces the sample (more than one of these)

• stage: platform where microscope slide is placed

• coarse and fine adjustment: focuses the view by moving the stage up and down

• condenser: condenses light through the sample

• diaphragm: opening of condenser

how do you properly use and store a microscope?

to carry a microscope

• hold the microscope at the arm, other hand holds microscope from the bottom

using a microscope

1. place sample on stage and begin with lowest magnification objective (10X)

2. focus with coarse focus, make it sharper/clearer with fine focus

3. turn to higher objective lens and refocus using fine focus

how do you focus a microscope on the 100X oil objective?

for the 100X objective lens, immersion oil is required to focus the loads of light that will scatter

1. turn the lens so that it is between 40X and 100X objective

2. add a drop of oil to the slide and turn to 100X (the lens will TOUCH the oil)

3. refocus using fine focus adjustment knob

what is a wet mount and what can you see in a wet mount?

wet mounts are used to view living cells in a wet environment

• place a drop of sample (broth, pond water) on a microscope slide

• place a COVERSLIP over the sample, then observe!

wet mounts view LIVING cells, helps to see motility of cells and how they interact within their natural environment

how do you calculate total magnification?

total magnification = ocular magnification x objective magnification

• ocular lens typically has 10X magnification

how do you prepare a heat fixed smear?

from a liquid culture:

1. use a loop to add a drop of culture to a slide

2. let the drop dry

3. pass the slide over flame 2-3 times

from a solid culture:

1. add a drop of water to the slide

2. add bacterial sample to drop

3. let drop dry

4. pass slide over flame 2-3 times

a smear is a sample of bacteria that are dried and heat fixed to a microscope slide so they don’t wash off during staining

what are the steps in gram staining?

1. add crystal violet (primary stain) to the smear, wait 1 minute, then rinse

2. add Gram’s iodine, the mordany, to the smear and rinse after a minute

3. add ethanol, the decolorizer, to the smear and rinse after 10 seconds

4. add safranin (secondary stain) to the smear and rinse the smear after 1 minute

5. blot the slide to remove excess liquid, then view the slide under the microscope

what are the steps in acid-fast staining?

1. add carbolfuchsin (primary stain) and let it sit for 20 minutes before rinsing

2. add acid alcohol decolorizer, let it sit for one minute, then rinse

3. add methylene blue (counterstain) and let it sit for 30 seconds before rinsing

4. remove excess liquid from the slide with blotting paper

what are the steps in endospore staining?

1. apply malachite green (primary stain) and a small strip of paper covering to the prepared smear

2. steam over heat source for 5 minutes (mordant)

3. rinse with distilled water (decolorizer)

4. apply safranin (secondary stain) to the smear, rinse after 30 seconds

5. rinse and blot dry

what are the steps in capsule staining?

1. add nigrosin stain (primary stain) to clean slide

2. use inoculating loop to aseptically mix bacterial sample with primary stain

3. create thin smear by dragging clean slide through sample

4. allow sample to air dry

   1. NO HEAT FIX, heat will destroy capsules

5. apply crystal violet (secondary stain) to smear, rinse after 1 minute

6. blot excess liquid off then place on microscope for observation

what does a gram-negative versus gram-positive look in a gram stain?

gram-negative organism: appears pink in stain, has thin peptidoglycan and an outer membrane

gram-positive organism: appears purple in stain, has thick peptidoglycan and no outer membrane

what do endospores look like in an endospore stain?

endospore = dormant state that is highly resistant to heat, dryness, etc.

• only some bacteria produce endospores

in endospore stain:

• endospores appear green, non-endospores appear pink

acid-fast staining colors

some bacteria contain mycolic acid in their cell walls, these are acid-fast-positive organisms, which appear pink

• non-acid-fast organisms appear blue

what do capsules look like in a capsule stain?

capsule stains are negative stains, so the capsules appear clear against the colored background and around the dyed cells

how do you perform a serial dilution?

solution is diluted multiple times to rapidly decrease concentration

1. pipette 1 mL of bacterial sample from stock tube into bottle A

2. shake bottle A

3. pipette 1 mL from bottle A to bottle B

4. shake bottle B

5. pipette 1 mL from bottle B into bottle C

6. shake bottle C

how do you calculate dilution and dilution factor?

dilution = (volume of solution or sample)/total final volume

1 mL of bacterial culture + 9 mL of water = dilution of 1/10

how do you calculate the CFU/mL of an original culture after a serial dilution?

CFU/mL of original sample = (# of colonies) x (reciprocal of dilution)

what are the steps in a standard plate count?

perform a serial dilution, then plate each dilution in the series, incubate the plates, and choose the plate with the countable number of colonies between 30 and 300 colonies

what is the difference between a pour plate and spread plate technique?

pour plate: sample is mixed into the agar before solidification, so there will be both subsurface and surface colonies

spread plate: sample is spread across the top of the solidified agar plate, so there will only be surface colonies

what are the phases in a growth curve and what is happening in each phase?

lag phase: bacteria are getting used to new environment, not a lot of growth

exponential phase: bacteria are growing quickly

stationary phase: running low on nutrients, growth slows

death phase: no nutrients, bacteria begin to die

what are the different oxygen requirements?

• strict/obligate aerobe: NEEDS oxygen to grow

• strict/obligate anaerobes: cannot live or growth in oxygen

• facultative anaerobe: can live with or without O_2, but prefers O₂

• microaerophile: likes some O₂

• aerotolerant: grows the same with or without O₂

how do the different oxygen requirements look on a thioglycolate tube?

• strict/obligate aerobe: growth only at top

• strict/obligate anaerobes: growth only at bottom of tube

• facultative anaerobe: growth throughout the FTM, more concentrated at the top

• microaerophile: growth at top but mostly slightly below the top

• aerotolerant: grows throughout tube, no area is more concentrated

what are the different temperature requirements?

psychrophile: LOVE the cold, 5 degrees C

pyschrotroph: 20 degrees C

mesophile: 37 degrees C is optimal

thermophile: 70 degrees C

extreme thermophile: 100 degrees C

what are the different pH requirements?

acidophile: optimum pH below 5.5

neutrophile: optimum pH near 7

alkalinophile: optimum pH above 8.0

what are the different salinity requirements?

halotolerant: CAN grow in high-salt environments

halophile: require high-salt environments above 13% concentration

osmophile: can grow in high-sugar environments

not tolerant: can only grow in near isotonic environments