Biosafety

NO FOOD OR DRINKS! Wash hands thoroughly Disinfect counters and work area Tie hair back Smock, apron, or lab coat optional Gloves and goggles optional Closed toed shoes required Eyewash in back sink. Safety shower/fire

extinguishers in back. Fire blanket in back above eyewash station.

• Always thoroughly wash hands upon entering and leaving lab.

• Keep work areas uncluttered and clean• Minimize splashes and aerosols• Disinfect work surfaces daily

• Sterilization• Disinfection

General Lab Use There will always be a

container of disinfectant on the back table for

disposal of contaminated material such as

slides, swabs, etc.

Disinfection:The use of a physical or chemical procedure to virtually eliminate all recognized pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects.

Sterilization: The use of physical or chemical procedures that destroy all microbial life forms, including highly resistant bacterial endospores. Autoclave: Pressurized steam at 15 psi and 121oC for an average of 20 min (10 – 40 min depending on bulk and load)

• Get me, do not pick up glass!• Wear disposable gloves • Cover large spill with paper towels and soak with

5% household bleach and allow to stand for at least 5 minutes

• Small spill - wipe with paper towel soaked in 5% bleach

• Discard contaminated towels in bleach container.• Wipe down the area with clean towels soaked in a

same dilution of household bleach

Don’t play with anything!!!!

Also don’t sleep…picture illustrates both.

It’s a micro lab. We work with bacteria. Don’t lie down on the

floor.

This was not ok in genetics.

It’s def not ok now. Don’t tell anybody about

this.

• Microscope (with accessories)• Inoculation loops• Source of flame (Bunsen burner)• Microscope slides and Cover slips• Gram staining kits • Petri dishes and proper growth media • Incubators • Autoclave (pressure cooker)

• Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant provided in lab.

Agar plates

Agar slants

Broth—liquid media

• General purpose• Enriched• Selective• Differential

• General purpose:• Supports growth of most non fastidious organisms• Tryptic Soy Agar

• Enriched • fastidious organisms—organisms that are pickier, more difficult

to grow.• Selective:

• Favors the growth of one type of microorganisms and inhibits the growth of others

• Saboraud Dextrose Agar (SDA)• Differential Media:

• Distinguishes between different groups of bacteria on the basis of biochemical characteristics

• Required for all microbiology preparations to assure that contaminants are not introduced.

• On a personal note, aseptic technique assures that infectious agents are not spread to you, fellow students, or the laboratory surfaces.

• The inoculating loop is usually used for making transfers of bacterial cultures (see next few slides for technique).

• Allow the loop to cool sufficiently so that any organisms to be tested will not be killed by the hot wire, but do not allow the loop to contact anything during the cooling period or contamination will result.

• Learn to remove and replace the caps or lids efficiently without setting them on the countertop or leaving the cover off too long.

• After the transfer is completed the loop must be sterilized again. Follow the procedure outlined on the following slides to prevent splattering of infectious materials.

• It is probably easier to work while sitting down. • Attention to details and practice will allow you to work both rapidly

and accurately.

In this lab, we will practice transferring bacteria from an agar slant and a broth culture to a TSA plate.

We will be using a technique called spot inoculation.

This will not give us isolated colonies. To get isolated colonies, we will do the streak plate

technique later in the semester.

1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer

2. Using the pinkie finger of your dominant hand twist the cap from the tube.  Hold in your pinkie and do not place it on the counter

3.     Pass the mouth of the culture tube across the flame 4.     Direct the already-flamed inoculating loop onto the slant and

remove some bacteria. You do not need much!!!! 5.     Flame the mouth of your culture tube again and replace the

cap.  Place it in your rack . 6.     Pick up the plate in your non dominant hand 8.     Move the loop in a small circle in one quadrant of the plate. 9. Put the plate back on its top. 10. Flame the loop.

1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer

2. Using the pinkie finger of your dominant hand twist the cap from the tube.  Hold in your pinkie and do not place it on the counter

3.     Pass the mouth of the culture tube across the flame. 4.     Direct the already-flamed inoculating loop into the broth. 5.     Flame the mouth of your culture tube again and replace

the cap.  Place it in your rack . 6.     Pick up the plate in your non dominant hand 8.     Move the loop in a small circle in one quadrant of the plate. 9. Put the plate back on its top. 10. Flame the loop.

Fig. 3.12.b

Fig. 3.12.c