POST LAB Flashcards by Miyaki Jan Lim

Why contrast is important in microscopy?

distinguish structures from the background
makes individual features or details visible

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How can contrast be achieved in using the microscope?

use of stains
adjust condenser diaphragm
in phase contrast microscope - generate constructive interference and reduce background light

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What is the relationship of wavelength and numerical aperture in getting a good resolving power?

There is a direct relationship between the NA and the resolving power of a microscope, while there is an inverse relationship b/w resolving power and wavelength

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How does oil increases resolution? Explain your answer

Oil has ↑ refractive index, which ↑ NA, resulting in an ↑ in resolving power. Simply, the use of oil allows better collection of light/ less diffraction

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Why does shorter wavelength light results to better resolution?

Shorter wavelength is less diffracted. Diffraction patterns may result in overlapping of the two points, making them unresolved.

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What are the advantages and/or disadvantages of using chemically defined and complex media?

Chemically defined media - chemical components are known/controlled

Adv. - controlled components, easily reproducible, less risk for contaminants.

DisAdv. - typically more expensive, limited microorganisms can grow

Complex media

Adv. - rich in minerals and nutrients that can allow a variety of microorganisms to grow, relatively less expensive as some components are derived from waste products

Disadv. - higher risk of contamination, lower reprodu

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bar or powdered form of dried agar or carrageenan, typically used as stabilizer, thickener or solidifying agent in cuisines/food industry

gulaman

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Agar vs. Gelatin

Agar ← seaweeds/macroalgae
Gelatin ← animal collagen

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No nutritional value thus no net effect in mcg growth
Most microorganisms cannot utilize it.
Stable at mesophilic temperature

Agar

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Blood vs. Chocolate Agar

Blood Agar - Growth factors are inside the cells (bacteria capable of hemolysis)
Chocolate Agar - Growth factors are in the medium

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What are the similarities and differences between enrichment and selective culture media?

Similarities: Both allows growth of a particular group of microorganism.
differences: Enrichment is for the proliferation of a typically low population species while selective is not intended for that

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BAP
Differential
Selective

Hemolytic properties
Non-hemolytic can still grow

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MSAP
Differential
Selective

Mannitol fermentation
Salt tolerants only

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MacConkey
Differential
Selective

Lactose fermentation
Inhibits gram (+)

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EMBA
Differential
Selective

Lactose fermentation
Inhibits gram (+)

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What do we usually use for labelling materials? What information do we usually write on the label?

Group number/ Section
Name of media
Date
Set-up or treatment
Inoculant/ Name of Microorganism

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Where do we usually place the label on the flask, test tube, Petri dish plates, and others?

Flask - dedicated white marking strip, avoid the graduations marks
Petri dishes - bottom plate
Tubes - 1” before the top to avoid burning when flamed but visible when placed in a rack

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Sheep’s blood in preparing blood agar plates? Any other animal blood that can be utilized instead? How about human blood?

✅ Sheep’s availability and sheep are large enough to extract enough blood for preparation
✅ Hemolysis is also very evident when sheep’s blood is used
✅ Horse blood is also a good alternative
✅ Human blood can pose higher risk for transmitting human infections, ethical concerns, poor isolation rates.

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process of eliminating all viable mcgs to remove contaminants that could affect results of experiments

sterilization

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Why do we need to sterilize culture media? What is the purpose of sterility testing?

Sterility testing ensures that the right conditions for sterility were achieved.

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How do we sterilize heat-sensitive culture media?

Filter sterilization using membrane filters with very small pores (0.2 microns)

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How long do we store culture media?

3-4 weeks in 2-8 °C

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Why do we incubate plated media upside down?

To prevent moisture leaking on the surface of the media which could spread colonies

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What do we do with contaminated plates? How about used plates?

Contaminated and used plates are decontaminated via steam sterilization and disposed

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Repeated clock streaking is good for making pure/axenic cultures since isolated/discrete colonies can be obtained.

Clock Streaking

Obtaining countable bacterial colonies, especially when done with serial dilution

Spread Plate

Sampling microbial load of ambient environment

Direct Plate Exposure

Obtaining a lawn of bacteria on a plate/confluent growth

Flood Plate

What will happen if... A non-well isolated colony is captured and grown on a fresh medium

Result in mixed culture, re-streaking is necessary to obtain pure culture.

What will happen if... Open a tube and not pass it over a flame

The tube might get contaminated and succeeding experiments using the same culture might also get contaminated as well

What will happen if... Media were not incubated during incubation

Water droplets might fall to the surface of your medium w/c are potential sources of contamination and might cause your colonies to spread

Aside from maintaining the adherence of the cells to the slide, what are the benefits of heat-fixing the cell?

✅Increases the permeability of the cell to the stain ✅Helps in the preservation of the specimen

f there are acidic and basic dyes, are there neutral dyes? Give examples and discuss the mechanism of their staining property.

✅Giemsa stain, eosin methylene blue, Leishman stain, Wright’s stain ✅Usually a combination of acidic and basic dye which can differentially stain different cellular structures

Utilizing different stains to simultaneously stain different structure

differential staining

example of differential staining

Gram staining, Acid-Fast staining, Endospore staining

Purpose of using steam in endospore staining?

Steam serves as the “mordant” to allow the malachite green to penetrate the endospore

What are the alternatives to steaming?

Heat fixing, using higher concentration of malachite green, increasing contact time

staining that detects the presence of acid fast bacteria from tissue samples.

Acid Fast staining

Other tissues can be decolorized by acids but some bacteria are resistant to this process due to the presence of ___________ in their cell wall. Thus, during the process the bacteria retains the primary stain (carbol fuschin) while other cells are stained by the secondary stain (methylene blue).

mycolic acids

temperatue set up results B. subtilis G. stearothermophilus

- 20 C no growth B. subtilis 37 C - optimal growth, 56 C - no or minimum growth G. stearothermophilus 56 C - optimal growth, 37 C - no or minimum growth

The effect of temperature was ____________, because when the tubes were placed in their optimum temperature, the bacteria were able to proliferate which indicates that the sub-optimal temperatures were only able to arrest their growth but not kill them.

bacteriostatic

pH set up results S. auerus

200 μl of 1N NaOH - 500 μl of 1N NaOH No growth 200 μl of 1N HCl + 500 μl of 1N HCl No growth Control (S. aureus only) ++++

Salt Concentration set up results S. aureus B. subtilis E. coli P. aeruginosa

S. aureus, halotolerant, while the rest can thrive in normal physiological saline only

Oxygen set up results B. subtilis E. coli

B. subtilis Thioglycolate - Growth was observed on the top of the medium. Pyrogallol KOH - No growth obligate aerobe E. coli Thioglycolate - Growth was observed all over but much concentrated at the top Pyrogallol KOH - +++ growth on the slant facultative anaerobe

Desiccation set up results B. subtilis E. coli

B. subtilis Immediately pour-plated ++++ Placed in a locker +++ E. coli Immediately pour-plated +++ Placed in a locker No growth

Which test organism was still able to grow despite losing moisture? What characteristic/s allows it to grow despite being desiccated?

B. subtilis was still able to grow even after desiccation, because it can revert to resistant endospore form. E. coli does not produce endospores, thus it cannot survive the loss of moisture.

Radiation set up results S. marcescens

S. marcescens Exposed to UV = No growth/decreased growth Not exposed to UV = ++++

Describe what happened to the S. marcescens exposed to UV. Explain the phenomenon behind this

UV radiation can damage the DNA of S. marcescens by creating thymine dimers resulting in its low/no growth after exposure

test for the Hydrolysis and fermentation of lactose to determine presence of lactase enzyme

Carbohydrate fermentation test using Lactose

Carbohydrate fermentation test using Lactose positive results negative results primary reagent/s

positive results Yellowing of phenol red negative results Phenol red not changing color primary reagent/s Lactose and phenol red indicator

test for the Hydrolysis of Casein (proteolysis) to determine ability to produce caseinase/proteases

Casein hydrolysis test

Casein hydrolysis test positive results negative results primary reagent/s

positive results Clear zones forming at the edges of colonies negative results Plate retaining its milky white color primary reagent/s Skim milk (contains milk proteins called casein)

test for the Hydrolysis of Casein (proteolysis) to determine ability to produce gelatinases /proteases

Gelatin Hydrolysis Test

process of Gelatin breaks down the protein to shorter polypeptides so it will not be able to tangle back and solidify.

Proteolysis

______ gives the plate its milky white color, When the protein is denatured through proteolysis, the milky white color will become clear.

Casein

_________ would lead to fermentation then acids will be produced resulting in the yellowing of phenol red

Hydrolysis of lactose

Gelatin Hydrolysis Test positive results negative results primary reagent/s

positive results Gelatin remains seminsolid/liquified even in cold temperature negative results Gelatin becomes solid when placed in cold temperature primary reagent/s Gelatin

test for the Ability to break down starch using amylases

Starch Hydrolysis

Starch Hydrolysis positive results negative results primary reagent/s

positive results No dark blue complex after addition of iodine negative results Production of dark blue complex after the addition of iodine primary reagent/s Starch and iodine

If ___________ is hydrolyzed/broken down, there will be no scratch to react with iodine. Production of dark complex means that ___________ is still intact.

starch

Why are there multiple positive result in Carbohydrate fermentation test?

Fermentation could either heterolactic or homolactic which generates different products. Heterolactic fermentation produces acids and carbon dioxide while homolactic will only produce acids

Why add the skim milk to the sterile NA and not include the Skim milk in the autoclaving of the NA?

Milk tends to coagulate when heated at high temperatures which can affect your results

Why specifically use skim milk, not full cream milk?

Skim milk contains significantly more casein and reduces the interference of fat to your results

Other biochemical tests (ie. Urease, Oxidase, Catalase, IMViC, etc) are also used for further phenotypic characterization of a bacteria. What are the advantages and disadvantages of biochemical characterizations for bacterial id? Practical uses?

- Advantages: inexpensive and easier to perform, characterize the specific strain/bacterial culture - Disadvantages: could be influence by many factors and results can be strain specific - Practical uses: microorganisms can be used as “factories” for the production of certain metabolites/substances if we are able to determine their biochemical properties.

3 common biochemical kits manual identification of gram negative, gram positive and yeast

API Biomerieux

3 common biochemical kits bacteria, yeast and filamentous fungi, automated high throughput analysis

BIOLOG

3 common biochemical kits typically used in healthcare settings

VITEK

In general, what is/are the advantages and disadvantages/limitations of biochemical diagnostic kits?

Advantages: easier to perform, reliable results, QA and QC, reproducibility, high-throughput - Disadvantages: more expensive, system could have difficulty in distinguishing microorganisms

Possible observations on the bacterial and disinfectant controls (assuming no error was committed, and all steps were done aseptically)

Disinfectant control - no growth Bacterial controls - exhibit growth

disinfectant; basis of mechanism of action oxidizes proteins, nucleotides and fatty acids effective against endospore

Iodine

disinfectant; basis of mechanism of action coagulates/denatures proteins not effective against endospore

Ethyl alcohol and isopropyl

disinfectant; basis of mechanism of action oxidation forming free radicals which damages cell components effective against endospore

Hydrogen peroxide

disinfectant; basis of mechanism of action oxidation, lipid destruction, protein denaturation effective against endospore, not effective against bacterial spores and prions

Hypochlorite

Agencies related to health care, such as U.S. Centers for Disease Control (CDC) and World Health Organization (WHO), recommend that cleaning should be done before disinfection. Explain.

Dirt and impurities makes can make it harder for the disinfectant to penetrate the bacteria reducing its effectiveness. Cleaning ensures that dirt and impurities are removed first prior to disinfection