Lab practical 1

What are the correct order of steps for a broth to agar plate transfer?

1. Sterilize the inoculation tools through the hottest part of the flame, the cone

2. Take the lid off the liquid culture

3. Pass the mouth of the liquid culture through the flame

4. dip the loop through the liquid culture

5. Pass the mouth of the liquid culture through the flame

6. Transfer the bacteria to the plate, lift the lid of the plate and use it as a shield to prevent contaminants from falling

7. Pass the loop through the flame

What are the correct order of steps for a broth to broth transfer?

1. Pass the inoculation loop through the flame

2. Take the lid off the liquid culture bottle

3. Pass the mouth of the liquid culture bottle through the flame

4. Dip the loop into the liquid culture

5. Pass the mouth through the flame again and replace the lid

6. Take the tube of the sterile broth tube and pass the mouth through the flame

7. Dip the loop into the sterile broth tube

8. Pass the mouth of the tube through the flame and replace the lid

9. Flame the loop to sterilize

What is the proper use of a bunsen burner?

good question

What are the 4 requirements for BSL 1?

• Lab work is performed at a bench

• PPE includes lab coat, apron, goggles, and gloves worn as needed

• Sink is available for hand washing

• Lab room should be separated with a door from the rest of the buliding

What are the 5 requirements for BSL 2?

All requirements for BSL 1 in addition to:

• Access to these labs is restricted to approved personnel only

• Labs have self closing doors

• Eye wash stations are available

• An autoclave or equal method of sterilization is available

What are the 5 requirements for BSL 3?

All requirements for BSL 1 and two plus:

• Personnel must have necessary vaccinations

• Work is performed in a biohazard safety cabinet

• Air supply to the lab room must be drawn from a clean supply and cannot be recirculated

• Lab room must have two self closing and lockable doors

What are the requirements for BSL 4?

All requirements for BSL 1, 2, and 3 plus:

• Personnel must change clothing and shower before and after entering these labs

• Biosafety cabinets must be a special level III cabinet

• The laboratory is located in a separate building or in an isolated and restricted zone of the buliding

• The laboratory has dedicated supply and exhaust air, as well as vacuum lines and decontamination systems

What microorganisms are used in BSL 1?

Escherichia coli

What microorganisms are used in BSL 2?

Staphylococcus aureus

What microorganisms are used in BSL 3?

Mycobacterium tuberculosis

What microorganisms are used in BSL 4?

Ebola and Marburg viruses

What is the definition of sterile?

Completely free from living microorganisms

What is the definition of aseptic technique?

A combination of protocols that collectively maintain sterility, or asepsis, to prevent contamination of a patient or surface with microbes and infectious agents.

How do you calculate total magnification?

Ocular lens magnification * objective lens magnification

What are all the parts of a brightfield microscope?

Ocular lenses

Revolving nose piece

Objective lenses

Larger knob

Smaller knob

Illuminator

Diaphragm

Condenser

Stage

What is the ocular lens?

The top lens you look through

What is the rotating nosepiece?

It holds multiple objective lenses

What are objective lenses?

They collect light passing through the sample and magnify it to create a clear image

What is the illuminator?

A high-intensity bulb below the stage

What is the stage?

Platform of the microscope

What is the condenser lens?

It focuses light rays on the specimen to maximize illumination

What is the larger knob? What does it do?

Coarse adjustment, quickly moves the microscope stage up or down in large steps.

What is the smaller knob? What does it do?

Fine focus knob, it is used to sharpen the image of the sample and focus the image

What objective lenses can the coarse focusing knob be used for?

4x or 10x

What lenses can the fine focusing knob be used for?

40x or 100x

What is the rheostat?

A dimmer switch that controls the intensity of the illuminator

What is the diaphragm?

It controls illumination and is located beneath the stage, just above the light source

What magnification can immersion oil be used on?

100x objective lens

What is the definition of resolution?

The ability to tell that two separate points or objects are separate

Take a quiz on brightfield microscope parts. now.

okay!

What factors affect resolution?

Wavelength and numerical aperture

What is the procedure for fixing bacteria to a slide?

1. Place a tiny drop of water on a clean glass slide

2. Use a sterile loop to mix the specimen onto the water, spreading it thin

3. Let it dry

4. Pass the dried slide through a flame three or four times

What is the procedure of gram stain method?

1. Heat fix the slide with the specimen by passing it over a heat source several times very quickly

2. Flood the heat-fixed bacteria with crystal violet for 60 seconds.

3. Rinse with water

4. Add iodine for 60 seconds

5. Rinse with water

6. Rinse with decolorizer, such as alcohol or acetone for 5-10 seconds

7. Counterstain with safranin for 45 seconds

8. Rinse with water

How do you interpret gram stain results?

If the bacteria are purple, it is positive, if the bacteria are pink, it is negative

What is the procedure of acid fast stain method?

1. Apply carbolfuschin for 30 seconds

2. Heat fix the cells to the slide using a flame

3. Decolorize with acid alcohol for 15-20 seconds

4. Apply methylene blue for 30 seconds then rinse excess stain

How do you interpret acid fast stain results?

Pink or red with be acid fast positive, blue will be acid fast negative.

What is the procedure of endospore stain method?

1. Air dry and heat fix the specimen on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide

2. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required. The slide can also be steamed over a contained of boiling water.

3. Wash the slide in tap water

4. Counterstain with safranin for 30 seconds. Wash with tap water and blot dry.

How do you interpret endospore stain results?

Endospores are bright green and vegetative cells are brownish red to pink.

What are the two streak plate methods of isolation?

T- streak method and quad-streak method

What is the spread plate method of isolation?

A lab technique used to separate and count live organisms in a liquid sample. Specimen are placed onto a solid agar plate and spread evenly.

What is the colony forming unit? What is the formula?

A measurement of live, active microorganisms in a sample.

(Number of colonies x dilution factor)/volume plated

What is the T-streak method?

Three sections are created on the plate, separated by a T shape.

What is the quad streak method?

An agar plate is split into four sections