Lab Practical Study Guide

it's just one more exam darling

What level of BSL precaution do we follow?

BSL-2

why do we wear lab coat, closed toed shoes, and have hair tied back

prevent contaminating the working space and for our own protection

when should we not wear gloves?

around a flame; they could melt and injure the hands

when is eye protection worn?

staining and using chemical reagents

where do used test tubes, media, and other contaminated glass re-useable (slides, flasks) go?

red autoclave cart

where do contaminated disposables (gloves, paper towels) and ALL agar plates go

into a biohazard bin

where do uncontaminated gloves and paper towels go

into regular trash cans

where do contaminated sharps (broken wet-mount slides, broken tubes with culture) go

in the biohazard sharps bin after dousing with 70% EtOH

where do clean uncontaminated sharps (razor blades, broken glass) go

into regular sharps bin

what should always be on a label

GREEN
Lab section (S2)
Date you inoculated (month/day)
Incubation temperature (in Celsius)
Culture source
Species if known (never write out full genus name; B. subtilis)
Genus if known, but species not known (ex. Staphylococcus sp. )
If neither species or genus is unknown (name of environmental source)
SOMETIMES include:
First name ONLY
Experimental conditions (streak #, controls)

what should be on the label for microscope slides

source, stain type, date

What is it and what are the pros and cons

TSA broth
Pros: Grows bacteria in large quantities, quickly, under uniform conditions
Cons: Easily contaminated

what are the different types of solid media

slant, deep, and plate

what is the function of agar

Agar: the gelling agent used in most solid media
Cheap, sustainable, no nutritive value (few bacteria can digest it)

what is it and what is it used for

solid Petri dish media for isolation of pure cultures and various tests

What is it and what are the pros and cons

TSA slant: solid test tube media for long-term storage of pure cultures and biochemical tests

Pro over broth: easy to catch contamination; look for unexpected growth off the S-path
Pro over plates: smaller surface for air exchange, so less likely to be contaminated and do not dry out as quickly
Con: not enough room to isolate pure culture; only used when pure culture has been isolated

What is it and what is it used for

TSA deeps: solid test tube media for long-term storage of pure anaerobic cultures and oxygen requirement tests (stabs)

what is defined/minimal/synthetic media

media that contains nutrient in pure chemical form and does not contain any plant or animal tissue; the exact chemical composition of the media is known

what is complex/undefined media

complex/undefined media: media that contains nutrients from plants or animal extracts; the exact chemical formula is not unknown

what are non-fastidious microorganisms

microorganisms that grow on general purpose media like TSA (our lab stuff)

Fastidious microorganisms

microorganisms that require specific nutrients or growth factors that are not found in general purpose growth media (pathogenic)

Autoclave

a machine that uses pressurized steam at high temperatures for a defined period of time; 121 degrees Celsius with steam pressure of 15 psi for 15 minutes

do long autoclave times increase sterilization

no, it might even burn and caramelize the sugars in the media

Why do we autoclave media, equipment

media ingredients/equipment are not sterile and will be contaminated with microbes from surrounding air, glassware, spatulas, and so on

Why do we autoclave biohazard

to kill any remaining microbes in the bags, safer disposal

Why autoclave and not boil?

Autoclaving can generate higher temperatures where microbes that are boiling-resistant cannot survive

what shape is this

spindle

what shape is this

filamentous; usually fungi

what are the different types of whole colony forms

circular, spindle, punctiform, irregular, wrinkled, filamentous, rhizoidal

what are the different types of colony elevation

flat, raised, convex, umbonate, convex with papilate surface

what are the different types of colony margin forms

entire, undulate, lobate, crenate, serrate, filamentous, rhizoidal

what kind of surfaces have high microbial load and diversity

soft, porous, natural material wha

what kinds of surfaces have lower microbial load and diversity

Hard, metal, non-porous, man-made surfaces

what are basic experimental design with control and hypothesis

Ask a scientific question
Form a hypothesis
Design an experiment to test your hypothesis
Collect results and analyze data
Draw a conclusion to support or not support your hypothesis

how to do a T-streak

a T-Streak as we taught it
Incinerator for 5 seconds, brand loop for 5 seconds, touch center of the colony (don’t scoop)
Streak outside to inside, incinerator for 5 seconds, turn the plate, brand loop into agar to cool, 2nd streak crosses 1st streak, incinerator for 5 seconds, brand loop into agar to cool, 3rd streak crosses 2nd streak, incinerator for 5 seconds, set sterile loop on the bench

what is this

turbid liquid culture

what is turbidity

microbes grow throughout the medius, making it cloudy

WWW: if there's no growth

the initial culture was dead or you killed the inoculum with a hot loop, or the loop did not touch the inoculum

what is Aseptic transfer:

the prevention of contamination of the culture and the microbiologist with unwanted microorganisms from the environment or other cultures

what is the Purpose of aseptic methods:

maintaining stock cultures and transferring pure cultures from one vessel to another

what is the purpose of Broth inoculation:

impossible to tell by eye if there’s a single species, must be determined microscopically; good for growing up pure cultures or cultures quickly

what is the purpose of slant inoculation

inoculation of a “S” patten up the agar surface; great for pure cultures with lower risk of contamination

what is isolation streaking

for obtaining pure culture from mixture of the environment; used to keep pure cultures alive and pure (subculturing); necessary for pure culture but careful of contamination

what is the only way you can confirm pure culture

checking microscopically for the same cell morphology and gram type

WWW culture transfers

WWW: don’t shake loop around, don’t leave plates open, don’t go back for more inoculum

purpose of broth to broth

grow cultures more rapidly

purpose of plate to plate

pure culture isolation

what are the four objective lenses

4x, 10x, 40x, 100xw

what are the two lenses

ocular len and objective len

a microscope that is ready to be put away

Set the scope to 4x BEFORE removing your slide
Lower the stage
Clean all objective lenses and ocular lenses
Wrap the power cord around the base
Replace dust cover

what is it and what does it do

Condenser: collects light; moves up and down to focus the light on the specimen

what is it and what does it do

Iris diaphragm: located between the lamp and the condenser, controls the amount of light that passes through the specimen

what is it

Objective lens: (4x, 10x, 40x, 100x) lenses closest to the specimen

what is it

ocular lens: eyepiece

how to calculate total magnification

Total magnification: 10x (ocular lens) times the objective lens

what mag to determine size

1000x

how to determine size

1 ocular unit is 10 microns (ocular units), one tick mark is 1 micron (always report in microns!!)
diameter/width and length of a cell
Might have to provide with a range for the length

Why do we need immersion oil to observe bacteria?

Resolving power: ability to distinguish two adjacent points as distinct and separate
Numerical aperture (NA): the cone of light that enters the lens; short wavelength is the best
Immersion oil improves the numerical aperture and hence the resolving power of the lens; it has a refractive index similar to glass

label this microscope

left
1. ocular lens
2. nosebridge
3. objective collar/wheel
4. objective lens
5. stage clips
6. stage
7. iris diaphragm
8. condenser
9. light source

right
1. NA
2. arm
3. stage
4. coarse knob
5. fine knob
6. stage control
7. base
8. light intensity dial
9. power

what stain is this

Crystal Violet: gram positive

what stain is this

Safranin: gram negative

what stain is this

Malachite green: endospores

what stain is this

Methylene blue: acid-fast stain

Prokaryotic vs. Eukaryotic cells

left: eukaryotes (HUGE) right: prokaryotes (smol)

Rods vs. Cocci

NOTE: short rods can be mistaken for cocci; gram negative cocci are rare, it's probably a dehydrated rod

Pairs vs. Tetrads

chains vs. single

How do you make a simple stain? What can go wrong?

smear a single-cell thick film on top of the slide (broth or water/solid), smear is air dried and heat fixed, slide is flooded with stain then rinsed with water
WWW: did not heat dry properly so cells washed off with the water, aimed the water at the cells
NOTE: timing isn’t that big of a deal with simple stains

what is a simple stain

Simple stain: a single stain is used, all cells and structures staint he same color

What is a positive vs. negative stain?

Positive stain: the stain attracts to the cells
Negative stain: the stain stains the background of the slide

Cheek swab -- differentiate prokaryotic vs. eukaryotic cells.

Eukaryotes (10 - 100 microns) are much larger than prokaryotic cells (0.1 - 5 microns)

Be able to heat fix and stain a sample of bacteria

Label: source, first name, date, simple, stain abbreviation (CV, Saf, MG, MB)
Smears: solid media (drop of water, spread over slide), liquid media (one drop, spread over slide)
Set hot plate to 3 and warm for at least a minute
Stain time:
Crystal violet: 10 seconds
safranin/malachite green: 30 seconds
Methylene blue: 60 seconds or longer
Rinse with DI water, aim for the labeled part
Blot dry with bibulous paper

crystal violet stain time

10 seconds

safranin/malachite green stain time

30 seconds

methylene blue stain time

60 seconds or longer

Be able to name and recognize the 4 simple stains we used

Crystal Violet, Safranin, Methylene Blue, Malachite Green

Be able to tell yeast and bacteria apart.

Yeast are huge!! Bacteria are tiny

what is it

Rods: hot-dog-shaped
NOTE: gram-negative coco are rare; you probably have a short gram-negative rod

what is it

Cocci: spherical
Tetrads: groups of 4
Cuboidal: packets of 8
NOTE: can appear oval just before cell division

what is it

Irregular rods: dumbbell-shaped, club shaped, cells can change shape over time

what is it

Vibrio: curve rods like a comma

what is it

Spiral: curly like a corkscrew

what is it

Spirochetes: spiral-shaped bacteria with many, many turns

what is it

Pleomorphic: change shape over time, making X’s, Y’s and other irregular shapes

trust wet mount or simple stain?

trust wet mount

Growth on a 12-well NaCl plate

NaCl tolerance starts from the lowest concentration of NaCl with growth to the highest concentration with growth
optimum NaCl concentration: the one with the most total growth

what is it

TSA deep: a confirmatory test for the Gas Pak anaerobic system

how to interpret results for oxygen deep

Aerobic: growth on the surface of the deep and top part of the line
Anaerobic: growth at the bottom of the line only
Facultative anaerobe: growth all the way down the stab line and growth on surface
Microaerophiles: no growth on top and growth in the middle (not top or bottom of line)

tolerant vs philic

Tolerant: doesn’t die
Philic: grows

what is strenotherms:

organisms that function over a narrow range of temperature

what is eurythermal:

microorganisms that grow over a wide range of temperatures (30 - 40 degrees)

what is stenothermals:

microorganisms that grow within a very narrow (

what is psychrophiles:

optimal growth less than 15 degrees Celsius; those that can grow below 15 but have a higher optimal temperature don’t count

what is mesophile:

optimal temperature between 20 to 40 degrees

what is thermophile:

optimal temperature between 45 and 80 degrees

what is hypterthermophile:

optimal temperature above 80 degrees

what is a halophiles:

bacteria that require high concentration of NaCl to grow

what is a hypertonic solution:

greater concentration of solutes than bacterium; water moves out of the bacterium into the hypertonic solution; cell shrinks and die

what is a hypotonic solution:

smaller concentration of solutes than bacterium; water moves into the bacterium; cell swells and bursts

what is a isotonic:

no water moves, bacteria can live