POST LAB Flashcards
Why contrast is important in microscopy?
distinguish structures from the background
makes individual features or details visible
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
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
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
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.
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
bar or powdered form of dried agar or carrageenan, typically used as stabilizer, thickener or solidifying agent in cuisines/food industry
gulaman
Agar vs. Gelatin
Agar ← seaweeds/macroalgae
Gelatin ← animal collagen
No nutritional value thus no net effect in mcg growth
Most microorganisms cannot utilize it.
Stable at mesophilic temperature
Agar
Blood vs. Chocolate Agar
Blood Agar - Growth factors are inside the cells (bacteria capable of hemolysis)
Chocolate Agar - Growth factors are in the medium
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
BAP
Differential
Selective
Hemolytic properties
Non-hemolytic can still grow
MSAP
Differential
Selective
Mannitol fermentation
Salt tolerants only
MacConkey
Differential
Selective
Lactose fermentation
Inhibits gram (+)
EMBA
Differential
Selective
Lactose fermentation
Inhibits gram (+)
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
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
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.
process of eliminating all viable mcgs to remove contaminants that could affect results of experiments
sterilization
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.
How do we sterilize heat-sensitive culture media?
Filter sterilization using membrane filters with very small pores (0.2 microns)
How long do we store culture media?
3-4 weeks in 2-8 °C
Why do we incubate plated media upside down?
To prevent moisture leaking on the surface of the media which could spread colonies
What do we do with contaminated plates? How about used plates?
Contaminated and used plates are decontaminated via steam sterilization and disposed
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
