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LBYBI13 > POST LAB > Flashcards

POST LAB Flashcards

1
Q

Why contrast is important in microscopy?

A

distinguish structures from the background
makes individual features or details visible

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2
Q

How can contrast be achieved in using the microscope?

A

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

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3
Q

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

A

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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4
Q

How does oil increases resolution? Explain your answer

A

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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5
Q

Why does shorter wavelength light results to better resolution?

A

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

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6
Q

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

A

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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7
Q

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

A

gulaman

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8
Q

Agar vs. Gelatin

A

Agar ← seaweeds/macroalgae
Gelatin ← animal collagen

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9
Q

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

A

Agar

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10
Q

Blood vs. Chocolate Agar

A

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

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11
Q

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

A

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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12
Q

BAP
Differential
Selective

A

Hemolytic properties
Non-hemolytic can still grow

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13
Q

MSAP
Differential
Selective

A

Mannitol fermentation
Salt tolerants only

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14
Q

MacConkey
Differential
Selective

A

Lactose fermentation
Inhibits gram (+)

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15
Q

EMBA
Differential
Selective

A

Lactose fermentation
Inhibits gram (+)

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16
Q

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

A

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

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17
Q

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

A

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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18
Q

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

A

✅ 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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19
Q

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

A

sterilization

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20
Q

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

A

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

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21
Q

How do we sterilize heat-sensitive culture media?

A

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

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22
Q

How long do we store culture media?

A

3-4 weeks in 2-8 °C

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23
Q

Why do we incubate plated media upside down?

A

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

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24
Q

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

A

Contaminated and used plates are decontaminated via steam sterilization and disposed

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25
Q

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

A

Clock Streaking

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26
Q

Obtaining countable bacterial colonies, especially when done with serial dilution

A

Spread Plate

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27
Q

Sampling microbial load of ambient environment

A

Direct Plate Exposure

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28
Q

Obtaining a lawn of bacteria on a plate/confluent growth

A

Flood Plate

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29
Q

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

A

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

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30
Q

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

A

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

31
Q

What will happen if…
Media were not incubated during incubation

A

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

32
Q

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

A

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

33
Q

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

A

✅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

34
Q

Utilizing different stains to simultaneously stain different structure

A

differential staining

35
Q

example of differential staining

A

Gram staining, Acid-Fast staining, Endospore staining

36
Q

Purpose of using steam in endospore staining?

A

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

37
Q

What are the alternatives to steaming?

A

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

38
Q

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

A

Acid Fast staining

39
Q

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).

A

mycolic acids

40
Q

temperatue set up results
B. subtilis
G. stearothermophilus

A
  • 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

41
Q

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.

A

bacteriostatic

42
Q

pH set up results
S. auerus

A

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) ++++

43
Q

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

A

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

44
Q

Oxygen set up results
B. subtilis
E. coli

A

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

45
Q

Desiccation set up results
B. subtilis
E. coli

A

B. subtilis
Immediately pour-plated
++++
Placed in a locker
+++

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

46
Q

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

A

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.

47
Q

Radiation set up results
S. marcescens

A

S. marcescens

Exposed to UV = No growth/decreased growth
Not exposed to UV = ++++

48
Q

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

A

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

49
Q

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

A

Carbohydrate fermentation test using Lactose

50
Q

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

A

positive results
Yellowing of phenol red

negative results
Phenol red not changing color

primary reagent/s
Lactose and phenol red indicator

51
Q

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

A

Casein hydrolysis test

52
Q

Casein hydrolysis test
positive results
negative results
primary reagent/s

A

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)

53
Q

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

A

Gelatin Hydrolysis Test

54
Q

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

A

Proteolysis

55
Q

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

A

Casein

56
Q

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

A

Hydrolysis of lactose

57
Q

Gelatin Hydrolysis Test
positive results
negative results
primary reagent/s

A

positive results
Gelatin remains seminsolid/liquified even in cold temperature

negative results
Gelatin becomes solid when placed in cold temperature

primary reagent/s
Gelatin

58
Q

test for the
Ability to break down starch using amylases

A

Starch Hydrolysis

59
Q

Starch Hydrolysis
positive results
negative results
primary reagent/s

A

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

60
Q

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

A

starch

61
Q

Why are there multiple positive result in Carbohydrate fermentation test?

A

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

62
Q

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

A

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

63
Q

Why specifically use skim milk, not full cream milk?

A

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

64
Q

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?

A
  • 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.
65
Q

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

A

API Biomerieux

66
Q

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

A

BIOLOG

67
Q

3 common biochemical kits
typically used in healthcare settings

A

VITEK

68
Q

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

A

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

69
Q

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

A

Disinfectant control - no growth Bacterial controls - exhibit growth

70
Q

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

effective against endospore

A

Iodine

71
Q

disinfectant; basis of mechanism of action
coagulates/denatures proteins

not effective against endospore

A

Ethyl alcohol and isopropyl

72
Q

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

effective against endospore

A

Hydrogen peroxide

73
Q

disinfectant; basis of mechanism of action
oxidation, lipid destruction, protein denaturation

effective against endospore, not effective against bacterial spores and prions

A

Hypochlorite

74
Q

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.

A

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

LBYBI13 (16 decks)

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