Lab Practical Study Guide!

Clinical Microbiology Laboratory!

Ocular Lens(10x) x Objective Lens(... ,10,40,100)

calculation of total magnification

resolution

minimum distance at which 2 distinct points of a specimen can be seen

parfocal

being able to refocus with minimal effort when changing to a different objective lens

Resolving power by keeping light rays from refracting

What is the purpose of using immersion oil

Lens paper

With what do we clean the lenses?

to isolate bacterial colonies

What is the purpose of dilution streaking?

heat loop and swab the broth with loop → streak at 90 degree angles the loop with the agar plate → 4th side should show single colonies

Know how to aseptically perform this technique

identical bacteria that look the same as their mother colony

What is a colony?

shape, size, color, surface appearance, and texture

What are colony characteristics?

pure: one bacteria
mixed: 2 or more bacteria on a plate; will have different shapes and sizes on agar plate
contaminated: bacteria from different source have contaminated the agar plate

What is the differences between a pure, mixed, and contaminated culture?

the lid can fall off the petri dish

Why label a plate on the bottom?

to lessen the risk of condensation compromising the colonies and lessen the risk of airborne contamination

Why incubate it in the inverted position?

to make them easily visible under microscope

Why do we stain cells?

adv: fixes bacteria on slide and preserves cells and kills microbes, disadvantage: inability to determine motility and distort cell shape and size

What are advantages and disadvantages to heat fixing slides?

basic: have positive charge so attract to cell wall and nuclei better than acid dyes: crystal violet, methylene blue, and Safranin
acidic: have negative charge and attach to positive components of cell); acid fuchsin

Know what basic and acidic dyes are and how they work

Bacillus, Coccus, or sprial

Know the basic shapes

Staphylo, Strepto, Diplo

Arrangements of cells

1 to 2 uM diameter and 5 to 10 uM length

bacteria typically fall within the μm range?

live bacteria to see if they are motile; steps: cover jelly on all 4 sides of the cover slip, place loop of suspension in center of cover slip, and then place concave slide on top of cover slip

Know how to prepare a hanging drop specimen

motility

What characteristic of the live bacterial specimen was observed using this technique?

to avoid drying out the drop and killing the bacteria

Why do you have to reduce the amount of light with the diaphragm to see bacteria in a hanging drop slide?

Gram-positive: contain teichoic acid, LPS not present, very susceptible to antibiotics and produce exotoxin and stain Purple
Gram-negative: LPS present, and produce exo and endotoxins, stain pink

Know the difference between a Gram-positive and Gram-negative cell wall

Primary stain: Crystal Violet
Mordant (fixes the dye): Iodine
Decolorizer agent: Ethyl Alcohol
Counter stain: Safranin

Know the basics of how to perform the Gram stain technique including the function of each component

not using morant-idoine properly

Under what circumstances might you get a false Gram-negative?

not using decolorizing agent: ethyl alcohol

Under what circumstances might you get a false Gram-positive result?

gram stain: differentiates gram positive and negative bacteria
acid-fast stain: differentiates mycobacteria (red) from non-mycobacteria (blue/green)

Why is the Gram stain (and acid-fast stain) considered to be a differential staining technique?

Carbolfuchsin (15 to 20 min) → Heat (3x hover over bunsen burner) → Acid Alcohol (20 sec) → Methylene Blue (60 seconds)

method for acid-fast staining.Know the staining procedure and function of each component

due to their lipid rich cell envelope

Why are mycobacteria acid-fast?

highly resistant to disinfectants and drying out

What advantages does this provide to the organism?

tuberculosis

What species cause important human diseases?

Primary stain: malachite green
Mordant: heat
Decolorizer: water
Counter stain: Safranin

The endospore-staining procedure was provided in a handout. Know the staining procedure and function of each component.

non-growing cell;dormant; green color

What are endospores?

growing cell; pink color

What are vegetative cells?

germination: the process by which the dormant spore is converted into a vegetative cell.
sporulation: the formation of spores

Define germination and sporulation?

bacillus and clostridium

What two genera characteristically produce endospores?

antrax, bolutlism, and tetanus

What human diseases discussed in lab are associated with spore-formers?

A steam-pressure sterilizer; uses high heat

What is an autoclave?

steam forms at 100 celsius, it’s temp inc. → inc. pressure; inc in pressure or temp= dec.(less time) to kill exposed heat-resistant endospores

How are steam, pressure, temperature, and time used in the sterilization process

hospital sterilization= 25 to 30 lb pressure, 133-135 celsius, and 5 to 25 min

What pressure, temperature, and time were used in the demonstrations?

if we can sterilize endospore cell walls(control) then we can sterilize everything else

Why is an endospore sample a valid sterilization control?

We did streaking technique. We mixed E. coli with each different disinfectant to see which one will kill the bacteria. Thick walls surrounding endospores are more resistant to adverse conditions than the vegetative bacterial cells.

Understand how you tested disinfectant (and antiseptic) activities on vegetative and endospore-forming bacteria in a time-dependent fashion. What was the overall outcome?

We labeled a blood agar plate and divided it in half to test before and after results. We use our fingers from tapping the before to then tapping the after section

How did you test the effectiveness of hand hygiene practices?

concentration of disinfectant, nature of material being disinfected {organic matter, biofilms}, pH/ temp, contact between disinfectant/ area to be disinfected, time of exposure, # of microbes, microbial characteristics

What are the principles of effective chemical disinfection?

Most resistant: Prions
Least resistant: viruses w/ lipid envelopes

Which types of organisms are most or least resistant to chemical biocides?

1.Sodium hypochlorite, ethyl alcohol: alcohols, determining of ethanol
2.Hydrogen peroxide: peroxygen, cleanse wounds
3.Lysol, Iodophor: halogens, skin disinfection/wound treatment
4.Antiseptic mouthwash, chlorhexidine gluconate: biguanide, surgical scrubs/ preoperative skin preparation

As for the quiz, you should know the disinfectants and antiseptics used in lab.

Avoid cross contamination of germs

In a clinical setting, why do you wash your hands before and after wearing gloves?

Infections acquired during the process of receiving health care that was not present during the time of admission.

What is a nosocomial infection?

It physically destroys germs/ removes germs and chemicals from skin.

Why is hand-washing an effective way to break the transmission of infection?

Microorganisms that are usually not found in the body

What are transient bacteria?

Transient flora colonizes the superficial layers of the skin, and is more amenable to removal by routine hand hygiene.

Are normal flora or transient bacteria easier to remove via hand-washing?

The test helps healthcare practitioners to determine which drugs are likely to be most effective in treating a person’s infection.

In a clinical setting, why is it important to perform antimicrobial susceptibility testing?

Take swab that has bacteria and swab a plate to create a uniform layer, divide plate into 4 quadrants, then place a disk in quadrants.

Understand the basics of how to perform the Kirby-Bauer disk diffusion test.

Appearance of bacterial colonies when all individual colonies on a dish agar plate merge to form a field of bacteria.

What is a bacterial lawn?

Measure the diameter of inhibition zones around the antimicrobial disk, measured in millimeters. To read resistance/ susceptibility: < 10mm: resistant, 11-15mm: intermediate, >16mm: sensitive

Know how to measure the diameter of the zone of inhibition as well as how to interpret the reading regarding resistance and susceptibility for a particular antibiotic.

Determine the minimum inhibitory concentrations of antimicrobial agents.

What is an E-test?

Place a continuous concentration gradient strip on blood agar plate, after you incubate it would show how much of ampicillin diffuses into the agar.

How did you determine the minimum inhibitory concentration (MIC)?

Explaining the meaning of something

What is the interpretation?

Plasmid play a central role as the vehicles for resistance gene capture/ their subsequent dissemination.

How did a plasmid confer resistance to ampicillin?

Have sterile nutrient broth, then add ampicillin to the first tube, then transfer to another tube, then transfer to the third, and continue this process till you fill all your tubes. Then transfer e.coli into the tube, mix content, then add the organism suspension to the antibiotic containing broth. You interpret the MIC by the qualitative categories (susceptible, intermediate, and resistant)

Know how to perform the “Broth Dilution Method” and how to interpret the MIC.

Growth controls help to check bacterial growth and sterility control is monitor/ control time, temp, pressure.

What are the purposes of the growth and sterility controls?

Cell wall, protein synthesis, nucleic acid synthesis.

What are antibiotic targets of the bacterial cell?

Limiting uptake of a drug, modification of a drug, inactivation of a drug.

What are the modes of resistance developed by bacteria?

Conjugation, transformation, transduction

Know the three mechanisms of horizontal gene transfer.

Cell wall synthesis, protein synthesis, DNA Gyrase, folic acid synthesis

What were the general modes of action of the antibiotics studied in lab?

1.Staphylococci: gram positive non-spore-forming spherical, appearance of “grape: clusters.

2.Streptococci: gram-positive facultative anaerobes, round shape, chain like (diplococci).

3.Pneumococci: gram-positive, lancet-shaped, chains.

4.Enterococci: gram-positive, spherical, short chains.

What are the Gram reaction, cell shape, and arrangements of these genera?

Catalase enables bacteria to metabolize harmful hydrogen peroxide and oxygen (bubbles).

Staphylococcus are catalase +, Streptococcus are catalase -

Know the catalase reaction and how this can differentiate between the two genera.

Staphylococcus aureus, Epidermidis, Saprophyticus.

What are the three medically important staphylococci studied in lab?

1. Staphylococcus aureus is an opportunistic pathogen that typically inhabits the skin and mucous membranes of the nose as a commensal.

2. Staphylococcus epidermidis Like S. aureus inhabits the skin and mucous membranes. Can cause biofilms to grow on plastic devices in the human body.

3. Staphylococcus saprophyticus is often associated with urinary tract infections in women.

now how the following selective / differential media and tests are used to distinguish between the species.

used as a selective media for the isolation of pathogenic Staphylococci.

What is Mannitol Salt Agar?

used to differentiate coagulase-negative staphylococci and identify the isolate as staphylococcus saprophyticus.

What is Hemolytic reactions as well as colony morphology on blood- agar Novobiocin disk test?

use to different potential pathogenic Staphylococci such as Staphylococcus aureus from other gram-positive catalase-positive cocci.

What is a Coagulase test?

used to check for certain antibodies or antigens in body fluids including saliva, urine, cerebrospinal fluid or blood.

What is a Latex Agglutination Test?

Streptococcus pyogenes, Agalactiae, Pneumoniae.

What are the three medically important streptococci studied in lab?

Yes, it is the normal flora of the mouth

Is viridans part of the normal flora?

The human intestines/ female genital tract.

Where do enterococci normally reside?

Opportunistic pathogen that can cause UTI, bacteremia, endocarditis.

What is their primary role in disease?

To remove O2 and increase CO2

Why was S. pneumoniae incubated in the candle jar?

Collect the throat culture by rubbing the sterile swab tip on the surface of one tonsils and swab in the culture medium, then incubate.

Know how to obtain a throat culture.

Alpha Hemolysis: Green-presence of biliverdin, by-product of the breakdown of hemoglobin (S. pneumoniae)

Beta Hemolysis: Yellow-complete lysis of RBCs. A clear zone appears around the bacteria (S. pyogenes)

Gamma Hemolysis: lack of hemolysis (E. faecalis)

Know how the following selective/differential media and tests are used to distinguish between species of these organisms.

a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not.

What is TAXO A (bacitracin)?

a differential test used to distinguish between organisms resistant to the antibiotic optochin and those not.

What is TAXO P (optochin)?

identify members of the group D streptococci. Enterococcus faecalis can grow in bile salt and hydrolyze esculin, forming a black slant.

What is Bile-esculin?

Add four drops of reagent A and B to the extraction tube, place specimen sample in test tube, place strip into solution then interpret your results

Steps of a Rapid Strep A test kit?

Gram-negative rods; Some normally inhabit the human intestine and are non-pathogenic while others are pathogens that are transmitted via the fecal-oral route.

Members of Enterobacteriaceae are?

glucose fermenters, nitrate reducers, and oxidase negative. Typically non-lactose fermenters are pathogenic while lactose fermenters are non-pathogenic.

Enterobacteriaceae members are?

...

Know the substrates, enzymes, products, and how to read a positive reaction.

A selective and differential medium to isolate gram - enteric bacteria and differentiate lactose-fermenting from non-lactose -fermenting gram - bacteria

Define MacConkey agar?

A selective and differential medium to recover gastrointestinal pathogens

What is Hektoen enteric (HE) agar ?

1. On one end, select colony with the needle
2. Gently pull inoculating wire through by twirling
3. Push wire in until you reach H2S-indole compartment; notice a notch in the wire
4. Break of wire
5. With the wire punch holes on the bottom of last eight compartments

Understand how to perform the Enterotube II System test to identify members of Enterobacteriaceae.

is a retrovirus that uses reverse transcriptase to convert ss RNA to dsDNA

What is HIV-1?

Recognized as a distinct disease in 1981, human immunodeficiency virus (HIV) is the causative agent.

What is AIDS?

...

Understand the stages of disease progression and signs and symptoms of HIV infection and AIDS discussed in lab.

HIV kills immune system cells that help the body fight infections and diseases.

*Know that individuals with AIDS typically succumb to opportunistic infections and/or rare cancers.

How does HIV challenge the immune system?

...

Understand how an indirect ELISA was used to detect the presence of anti-HIV IgG antibodies in hypothetical patient serum samples. Know the steps of the assay and their importance.

if the patient has not yet developed antibodies to HIV

Are there any limitations of the ELISA for HIV testing?

Know that alleles for blood types A and B are codominant while the allele for blood type O is recessive.

Blood Grouping

is an inherited protein found on the surface of red blood cells. a woman with Rh-negative blood is impregnated by a man with Rh-positive blood and conceives a fetus with Rh-positive blood, sometimes resulting in hemolysis.

What is the Rh factor? How is it implicated in erythroblastosis fetalis?

A -> B Antibodies
B -> A Antibodies
O -> All antibodies
AB -> No antibodies

For a person with a specific blood type, what antibodies is he producing?

Universal Donor: O -> A, B
A,B -> AB (Universal Recipient)

For someone with a specific blood type, who can she donate to or receive blood from?

Rh + -> receive + or -
Rh - -> receive ONLY -

For someone with a specific blood type, who can she donate to or receive blood from?