Microbiology lab practical 1

classify the organism based on the result shown in tube A

Bottom only, no oxygen and OBLIGATE ANAEROBE

Classify the organism based on the result shown in tube B

Throughout, oxygen preferred, FACULTATIVE ANAEROBE

Classify the organism based on the result shown in tube C

Top only, oxygen required, OBLIGATE AEROBE

Classify the organism based on the result shown in tube D

Slightly below surface, low oxygen, MICROAEROPHILE

Give the name of the media used in 2042 in the oxygen tolerance test

Fluid Thioglycollate medium (FTM)

Give the name of the indicator presenting media used in 2042 in the oxygen tolerance test

Resazurin

What is the name of the indicator and inhibitor in media MSA

Indicator- Phenol Red

Inhibitor- high concentration of sodium, 7.5%

What type of bacteria will grow in media MSA

Staphylococcus species

What is the name of the indicator and inhibitor in media macConkey

Indicator- neutral red

Inhibitors- Bile salts and crystal violet

What type of bacteria will grow in media macConkey

Gram negative bacteria like E.coli

What is the difference between alpha, beta, and gamma hemolysis

Alpha- partial breakdownof red blood cells, greenish or brownish discoloration around colonies

Beta- complete breakdown of red blood cells, clear transpsrent zone around the colonies.

Gamma- no hemolysisclear, no color change around the colonies

What type of hemolysis pattern is shown in media E

Gamma, no change

What is the name of the inhibitors in media CNA and what type of bacteria will grow there

Inhibitor- colistin and Nalidixic acid

Bacteria- gram positive bacteria

Name the organism (genus) used under the microscope

Organism- vorticella

Genus- protozoan ciliates

Nigrosine stains do not enter cells because both the stain and the cell membrane are ———- charged

Negatively

What is the purpose of using the negative staining technique

To observe cell shape, size and arrangement without heat fixing.

Why did Clostridium sporogenes grow in the anaerobic box but not in the air

Because it is an obligate anaerobe

Which organism will grow in the air and not in the anaerobic box

Obligate aerobes

Give 2 modes of action of antibiotic

Inhibition of cell wall synthesis

Inhibition of protein synthesis

What is the difference between bactericidal and bacteriostatic chemicals

Bactericidal- kills bacteria directly

Bacteriostatic- inhibits growth and reproduction

What is the name of the antibiotic susceptibility test? What media is used to do this testing

Name- Kirby-Bauer disk diffusion test

Media- Muller-Hilton Agar (MHA)

Which antibiotic should you use to treat the patient, the bacteria that is resistant to or susceptible to it.

Susceptible

How should biohazard materials be disposed of

In the biohazard container with orange bags

Point of burning inoculation loop or needle

To sterilize

Purpose of a Petri dish/plate

To isolate colonies

Nutrient agar slant

temporary storage of culture

Nutrient broth

propagation of culture

Autoclave

Temp- 121°C

Pressure- 15 psi

Time - 15 minute

Parameter- standard

Point of four ways to streak

To obtain a pure culture

colony morphology shape

-Regular

-irregular

-filamentous( fungi and mold)

Colony morphology size

-pint point

-small

-medium (E.coli)

-large (B.megatherium)

-extra large (B. Megatherium)

Cell morphology

bacillus and filamentous

cell morphology color

-tan

-light

-opec

CNA

colistin and nalidixic acid agar

Selective- Forgrain positive

MacConkey Agar

selective- gram negative

differential - lactose fermentation

ASA Agar

Selective- staph species

Differential- pseudomonas species (yellow or green fluorescent light).

Gram stain steps

-crystal violet- 1/2 minutes

-rinse with dI water

-iodine/mordant/-1/2 minutes

-rinse with dI water

-Decolorize- 50 seconds

-rinse with dI water

-Dry slides with bibulous paper

Gram stain color

Gram positive- purple, s.epidermis

Gram negative- pink, E.coli

Will E.coli grow at 37 degrees after being incubated 4 degree and why

Yes because E.coli becomes metabolically inactive but it not killed. When returned to 37 degree, it resumes back to normal.

Define optimal temperature. What is the difference between psychrophile and mesophile

Optimal temperature, at which an organism grows best and metabolic activities are most efficient.

Psychrophile grows 0-20 degree

Mesophile grows 20-45 degrees

Resolution

The ability to distinguish 2 separate close objects as separate

Parfocal

When you change the objectives, the image remains mostly in focus

Field of view

The circular visible area seen through the microscope

Magnification

The ratio of the image size to the actual size of the object

What is the name of the microscope used most in microbiology lab

Compound light

What is the total magnification when using the 100x objective lens

100 x 10 =1,000

What is the purpose of the oil while using the oil immersion oil

To increase resolution by reducing light refraction

Name the structure formed in dNA caused by UV exposure

Thymine dimers

2 disadvantages of using UV for sterilization

Poor penetration

Damaged by dust or organic matter

What is the enzyme that can repair UV damage

photolyase

At what wavelength is UV harmful to bacteria

Around 260 nm

Why did we cover half of the plate while putting bacteria under UV light

To create a control side

Which bacteria is more resistant against UV and why

Bacillus species because they form endospores which protests DNA from UV

Why did we use the 3 way swab technique and how did you do it

To achieve even bacterial distribution across the agar plate

-swab the plate in one direction

-rotate plate 60-90 degrees

-swab again and repeat 3 times

What is the name of the instrument that sterilizes

Autoclave

Why do we use indicators while autoclaving

To verify successful sterilization

identity term A and its function

Inoculating needle,used to isolate pure colonies and stab into agar tubes

Identify term B and its function

Flint striker, used to ignite the Bunsen burner

Indentify term C and its function

Glass rod/ spreader, used to evenly distribute liquid bacteria across an agar plate

Identify term D and its function

Vortex machine, used to mix contents in a tube

Acidophile

Organism that grows best when in an acidic environment. Below pH 6.0

Neutrophile

Microorganisms that grows best in a neutral environment. PH 6.5-7.5

basophiles/alkalinophiles

Microorganisms that grow best in basic condition. PH below 8.0

Which type of microorganism will grow at neutral pH

E.coli, staphylococcus aureus

Which type of microorganism will grow at acidic pH

Lactobacillus

Smear Preparation

Flame the loop, cool, loop full of broth culture

Place the drop on clean slide, spread evenly

Allow smear to air dry before heat fixing

Four important steps in preparing a smear from AGAR culture:

1. Place one drop of sterile water on a clean slide.

2. Flame loop, let it cool, then collect a small amount of bacteria from the agar plate.

3. Mix it gently into the water drop to create a thin suspension.

4. Air-dry completely before heat-fixing.

What will happen if you take too many cells during smear preparation for Gram staining?

The smear will be too thick, causing:

• Poor stain penetration

• Uneven decolorization

• Inaccurate Gram results (may appear Gram-variable)

What would happen if you don't heat-fix while smearing from bacteria on a plate?

The bacteria will wash off during the staining process, and the smear may not adhere to the slide.

What would happen if you didn't air-dry completely before heat-fixing

Remaining moisture will boil during heat-fixing, causing:

• Cell lysis (cells burst)

• Distorted morphology

Why is it required to stain a specimen for observation in a bright-field microscope

Because most microbes are transparent, staining adds contrast so cells and their structures can be clearly seen against the bright background.

What is the pore size of the membrane filter?

0.45 micrometers

What does EMB stand for?

Eosin Methylene Blue

Coliforms are gram ___ and ___ fermenting rods.

Gram-negative and lactose-fermenting rods.

Define commensalis

A type of symbiotic relationship where one organism benefits while the other is neither helped nor harmed.

ldentify the staining technique used on microscope "A"

Smear and gram staining

Define complex or rich media. Give an example

contains nutrients of unknown exact composition, usually derived from extracts of plants, animals, or yeast.

Example: Nutrient agar, Tryptic Soy Agar (TSA), or LB broth.

Define synthetic media. Give an example.

the chemical components and their exact concentrations are know

Example: Glucose salts broth or minimal salts agar.

Name the two constituents of media that are required for bacterial growth.

Carbon source

Nitrogen source

What is the concentration of agar in broth?

• Broth has no agar (0%).

At what temperature does agar solidify after it is dissolved?

Agar solidifies at around 40-45°C after melting at about 85-100°C.

Define Phototroph, Autotroph, Chemotroph, and Heterotroph

Phototroph- Uses light as an energy source.

Chemotroph- Uses chemical compounds (organic or inorganic) as an energy source.

Autotroph- Uses CO₂ (inorganic carbon) as its carbon source.

Heterotroph- Uses organic carbon (e.g., glucose) as its carbon source

What is the difference between pure and mixed culture?

mixed culture contains more than one type of microorganism while pure culture contains only one type of microorganism

What will happen if you don't flame the loop before taking the culture to the next quadrant for four-quadrant streaking?

You'll carry too many cells into the next quadrant.

Do we take culture for inoculation for every quadrant? Why or why not?

No.

We only take the initial inoculum from the culture for the first quadrant.

Then we flame the loop and drag bacteria from the previous quadrant to the next to dilute the cells gradually, allowing isolated colonies to form.

How do we incubate plates in the incubator?

Upside down

Endospore colors

Green

Malakai green

Steam opens up pores

B.megatarium (heat tolerant)

acid fast stain

Bacteria and other microorganisms that resist decolonization by acids after being stained, a trait due to their high lipid cell walls.

What is Negative stain for

Used to visualize unstained cells against a dark background

Osmolarity

Fusion of water from high or low concentration

Lichen

Positive relationship

Antiseptic

Lysol, alcohol, listerine

Hypertonic

Having a higher concentration of solute than another solution.

Isotonic

Having the same solute concentration as another solution.

Hypotonic

Having a lower concentration of solute than another solution

Oxygen tolerance tube

FTM tube

Microaerophile

Enterococcus

Facultative ph

PH=4.5

Neutral pH= 7

UV

Enterococcus, epidermis, B.megaaterium

MIC

Lowest concentration of an anti orbital agent that inhibits the visible growth of a microorganism in vitro