MCRO 251 Lab Practical

Parts of the Microscope

1. Eyepiece
2. Nose piece
3. objective lens
4. stage clips
5. stage
6. diaphragm
7. light source
8. base
9. arm
10. condenser lens
11. course adjustment knob
12. fine adjustment knob

The condenser on the microscope is essential for what?

High resolution using oil immersion.

Resolving Power

ability of a lens to distinguish two nearby points as distinct and separate.
= wavelength (.55 micrometers) / 2x the numerical aperture

Oil is only used with the ___ objective

100X (be sure to remove oil from the oil immersion lens with lens paper)

Why should you keep screw capped media slightly ajar?

To allow for gas exchange. Really important in tubes that are supposed to show sugar or protein metabolism

Aseptic Technique

Used to prevent cross-contamination between cultured materials and the outside surfaces/objects.

Gram Staining

differential stain that divides bacteria into two groups: negative and positive
G+ = purple (thick peptidoglycan layer and polysaccharide)
G- = pink (thin peptidoglycan layer surrounded by an outer membrane)

How many steps in a Gram Stain Procedure?

1. Crystal Violet (dye/stain)
2. Iodine (mordant, sets dye)
3. Alcohol (decolorizes)
4. Safranin (counter stain)

What is the chemical basis for Gram stain distinction?

Cell wall structure. Peptidoglycan and polysaccharide.

What species tend to decolorize more easily?

Staphylococcus spp. and old cultures

What is the objective of streak plating?

To produce well separated colonies of bacteria from a mixed suspension.

During streak plating, what might happen if you do not let your loop cool prior to streaking?

prevent growth by killing bacteria, ultimately leading to no separated colonies.

Why should you invert your plate prior to incubation?

Prevent condensation.

What type of charge to bacteria have on their surface??

Negative. This is important when using direct stain (contain positively charged color ion or chromogen). Use a negative stain for cells that do not absorb traditional basic dyes.

What type of stain is used for indirect (negative) staining?

acidic stain that results in a stained background. Use a negative stain for cells that do not absorb traditional basic dyes.

What type of stain do endospores accept? Is there a counterstain used?

primary stain (malachite green). Safranin is a counter stain used to stain the vegetative cell.

What are the advantages of using an acidic stain?

Cells do not have to be heat fixed, and they are easier to stain this way.

How do we stain capsules?

Stain the background with an acidic stain, and counterstain bacteria with basic stain. You should see a halo signifying the capsule.

What type of agar did we use for the handwashing lab?

Sheep blood agar and Sabouraud agar. (propagate fungi)

What are environmental factors that must be satisfactory for each organism?

pH, temperature, oxygen concentraion

Why can't some organisms tolerate any oxygen at all?

They cannot breakdown products of oxygen metabolism like superoxide and H2O2.

Facultative anaerobe

Grows with or without oxygen (3)


requires oxygen (1)


cannot grow in the presence of oxygen (2)


grows in reduced oxygen (4)

Aerotolerant anareobe

organisms, which cannot use oxygen for growth (5)


kill organisms on the surface of inanimate objects


used on human tissue to kill microorganisms

What are two factors that determine the effectiveness of a chemical agent?

concentration and length of time.

Cidal v Static

kill, inhibit

What are other factors that influence efficiency of disinfectants?

Type of microbial population environmental conditions
Blood, puss, and tissue fluids.

What would happen if you did not carefully drain excess fluid from your disc prior to placing it on the agar?

It could run all over the agar and into other areas.


naturally produced by microbes ex: penicillin


natural or chemically synthesized.

Kirby-Bauer technique

measures sensitivity of a test microorganism to a range of antibiotics. Use a Mueller-Hinton agar.

True/False: Mueller-Hinton is not an example of a controlled variable in anti-biotic susceptibility testing.


What should be found on the Mueller-Hinton agar plates after incubation

zones of clearing/inhibition if the organism is sensitive.

What factors influence the size of the zone of inhibition?

Degree of susceptibility, size of inoculum, incubation time, temperature, diffusion rate of antibiotic, and growth rate of organism.

How is resistance conferred?

By genes carried on plasmids that are transferred by conjugation from one bacterium to another.

What is the basic requirement of an antibiotic?

It must show selective toxicity, or be inhibitory to the pathogen without harming the host.

What is conjugation?

genetic material from a donor bacterial cell is transferred to a recipient cell.

Bacterial Conjugation Process

1. Donor cell attaches to a recipient cell with its pilus. The pilus draws the cells together.
2. The cells contact one another.
3. One strand of plasmid DNA transfers to the recipient.
4. The recipient synthesizes a complementary strand to become an F+ c

Bacterial Conjugation Hfr

1. F plasmid integrates into chromosome by recombination
2. Cells join via a conjugation pilus
3. Portion of F plasmid partially moves into the recipient cell trailing a strand of donor's DNA
4. Conjugation ends with pieces of F plasmid and donor DNA in r

What must a cell have to initiate conjugation

Fertility Factor

Is bacterial transformation vertical or horizontal?

Horizontal gene transfer. Naked DNA

Origin of transfer

starting point for transfer to begin

Given that Strain 1 carries the gene for streptomycin resistance on the bacterial chromosome and Strain 2 carries the gene for ampicillin resistant on a plasmid, what happened when we mixed the 2 strains? Which of the original strains was the donor? recip

Strain 2 is the donor (plasmid) and strain 1 is the recipient (chromosome). Strain 2 formed a sex pilus with S1 and transfered its plasmid over to S1 to create bacteria that is resistant to both streptomycin and ampicillin.

Selective Media

inhibits growth of one type of bacteria will permitting growth of another

Differential Media

does not inhibit one type but displays different characteristics of different bacteria.

How do you calculate the concentration of phage in the undiluted stock?

Multiply the number of plaques on a plate by dilution factor using:
PFU/mL= (# of plaques)x(dilution)/ V of phage plated
Dilution factor=1/dilution

What color do oxidase positive organisms turn?


What is the catalase test for?

Production of the enzyme catalase. In this test you drop plasma on glass slide then mix the organism in and clumps are visible with staph and S. eppidermidis should just be milky

What are the two major enzyme systems involved in electron transport?

cytochromes and flavoprotein oxidases

True/False: Anaerobes generally have catalase enzyme


For what is the catalase test most often used for?

Used to differentiate Gram positive cocci from one another: staphylococci (clumps on glass slide) and streptococci

What test is used to differentiate pathogenic S. aureus from non-pathogenic staphylococci?


What does the enzyme coagulase do?

causes blood plasma to clot by converting fibrinogen to fibrin.

Which organism is coagulase positive?

S. aureus

What are the two most likely groups to be a Gram positive coccus?

Staphylococci and streptococci

What are the demonstrations done to distinguish catalase negative streptococci?

A and P discs, Bile esculin

Tell me about A Disc-Bacitracin

differentiates group A strep and beta-hemolytic non-group A strep based on sensitivity to bacitracin. If zone of inhibition grows around the disc after incubation, identification of group A strep is made.

Tell me about P disc- optochin

differentiates Strep pneumoniae from other alpha hemolytic streptococci based on sensitivity to optochin. If zone of inhibiton grows around disc, the organism is assumed to be S. pneumoniae.

What is bile esculin performed for?

Lancefield Group D streptococci can be distinguished from non-hemolytic cocci. E. faecalis can hydrolize esculin in the presence of bile to produce end product that reacts with iron, turning the slant black.

What two things can ELISA detect?

antigen or antibody

What do we look for in a direct ELISA?

presence of antigen in patient sample.

What do we look for in an indirect ELISA?

presence of specific antibody in patient sample.

True/False: A higher concentration of patient antibody results in a higher number of antigen:antibody pairings bound to the wall of the well.


Where does the color change in ELISA come from?

After rinsing the unbound secondary antibody from the wells, we add the enzyme substrate. When it binds, it will react with the substrate to produce color change.

What is the agglutination reaction?

particulate antigens and specific antibody directed against them result in the creation of antigen-antibody complexes

Blood Typing

1. If RBCs containing non-host antigens are mixed with antiserum a visible clumping can be seen. This clumping is the result of cross-linking between antigen and antibody
2. If the antibodies are not specific for the antigens and cannot bind to them; no l

Group O blood

universal donor; it contains neither A nor B antigens does contain antibodies to both A and B

Group AB blood

Universal recipients; contain no antibodies to agglutinate the donor's blood cells

Anti-A antiserum

O: -
A: +
AB: +

Anti-B antiseurm

O: -
A: -
AB: +

Triple Sugar Iron Agar (TSI)

identifies gram-negative enteric bacilli (enterobacteriaceae)
Tests for:
1. the fermentation of glucose, lactose or sucrose
2. the production of hydrogen sulfide (H2S) black
3. the production of gas (bubbles)

TSI results

1. A/A (yellow/yellow): acid slant, acid butt- ferments lactose and/or sucrose, in addition to glucose
2. K/A (red/yellow): alkaline slant, acid but - ferments glucose only
3. K/K (red/red): alkaline slant, alkaline but- none of the sugars are fermented

Citrate Utilization Medium

The pH indicators, bromothymol blue, is green at neutral pH and turns blue when the medium becomes alkaline. If a microorganism can use citrate, CO2 will be an end product and upon its release, the combination with sodium ions and water will result in the

Motility-Indole-Ornithine Decarboxylase Medium (MIO)

Differential - used to identify members of the Enterobacteriacease
Motility - non-motile grow only along the stab; motile migrate throughout causing the agar to appear turbid
ODC - used to determine the ability of an organism to decarboxylate ornithine to

Urease Test Agar

Separates Proteus from other Gram-negative bacteria that do not ferment lactose. Proteus produces the enzyme urease, which splits urea to form ammonia and CO2. Urea agar slant contain pheno red indicator as a growth medium. Ammonia accumulation turns the

different forms of hemolysis of blood agar

beta-completely lysed/clear
gamma-nothing happened no lysing

oxidase test

Neisseria spp and Pseudomonas spp are pos for this test and allow us to seperate them from enteric bacteria (which are negative)
positive organisms produce cytochrome oxidase-- oxidize artifical electron donor and when drop of test reagent is placed on fi