General Microbiology LaboratoryGeneral Microbiology Laboratory

Sterile Technique & Pure Culture ConceptSterile Technique & Pure Culture Concept

Why?

To protect your self from contact with bio hazards

To protect your sample from become contaminated

To protect others in the lab

In working with microorganisms, we must have a method of transferring growing organisms from a pure culture to a sterile medium without introducing any unwanted outside contaminants.

This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

For the most part, bacterial physiology only can be studied in pure cultures.

The best way to obtain a pure culture is to start with a single bacterial cell.

This cell then divides quickly, and may produce millions of cells within 24 hours.

A single unwanted contaminant cell can do the same thing in an otherwise pure culture, making the culture useless.

A. ASEPTIC TECHNIQUEA. ASEPTIC TECHNIQUE

FirstFirst

The most commonly used device for moving bacteria is the inoculating loop.

This is simply a piece of nichrome (an alloy of nickel and chromium) or platinum wire with a loop at one end and a handle at the other.

A similar instrument is the inoculating needle, essentially the same as the loop, but with just a straight wire

Leave the Bunsen burner on during your work

Sterilize both instruments by holding the wire portions in a flame until they glow red. The instruments should be allowed to cool in the air for 10-20 seconds before using them to avoid killing the inoculum.

In this way all contaminants on the wire are incinerated.

Do not blow on the instruments to cool them Do not touch the instruments to agar to cool

themDo not lay the loop down once it is sterilized

or it may again become contaminated.

The procedure for aseptically transferThe procedure for aseptically transfer

1. Flame the loop.2. Without setting the loop down, open the first

culture tube and flame the mouth (why) . Do not set the cap on the bench. The cap should be held in the same hand as the loop.

3. Insert the loop into the culture medium, then withdraw it.

4. Flame the mouth of the first culture tube again, and replace the cap.

5. Open the second culture tube and flame the mouth. Do not set the cap on the bench. The cap should be held in the same hand as the loop.

6. Insert the loop into the second culture tube and spread the culture suspension (on the loop) inoculum into/onto the second culture medium.

7. Flame the mouth of the second culture tube, then replace the cap.

8. Flame the loop and set on the bench.9. When in doubt about the sterility of an

instrument or container, sterilize it.

RememberRemember

BacteriaBacteria1. Are everywhere!2. On every surface of the body3. Including digestive tract4. Harmless5. Beneficial6. Pathogenic7. Absorb nutrients and release toxins that damage cells and tissues.8. Bacterial toxins can cause disease even when bacteria

are destroyed9. Bacteria are Prokaryotes

Five Basic Techniques of Culturing

1. Inoculate

2. Incubate

3. Isolation

4. Examination

5. Identification

Pure Culture ConceptPure Culture Concept

Attempts to identify bacteria in a clinical sample cannot be done unless isolated colonies are used.

To obtain well-isolated colonies, it is essential to disperse the inoculum (sample) on the surface of an enriched agar plate so that individual bacteria are well separated from each other.

Contaminants = other microorganisms present in the sample Isolated colois= a population of millions of cells that are

identical and are descendent from a single founder cell Stock Culture = a culture that already contains cells. It is used a source of cells from which to inoculate new

cultures. culture medium: rich/selective

• growth inhibitors• liquid/solid • temperature• source of energy• sources of carbon, nitrogen, ...

Aseptic technique:• sterilization of medium and equipment• proper handling

Necessary equipmentNecessary equipment

ProcedureProcedure

1. With the loop, spread the inoculum back and forth across the upper 1/4 of the plate, keeping the lines of inoculation very close together (area 1 in figure below).

2. Isolated colonies are not expected in this area. Do not use strong pressure, which will break the surface of the agar. Use the end of the loop, not its side when streaking. Dispose of the loop in the biohazard bucket on the bench.

3. Turn plate approximately 90oC. Streak the plate as indicated in the figure (area 2) across about 1/4 of the plate. Dispose of the loop.

4. Repeat step 2 one or two times more.5. In area 3 and/or 4 single colonies should appear.6. Label plates on the bottom and incubate inverted at 37oC.

Note: Lids on test tubes are loose. Always hold the glass test tube (not the lid)

when carrying them.

StreakingStreaking

FORMS OF CULTURE MEDIAFORMS OF CULTURE MEDIA

1. Broth tube: are tubes containing a liquid medium. A typical nutrient containing broth medium such as Trypticase Soy broth , nutrient broth After incubation, growth (development of many cells from a few cells) may be observed as one or a combination of three forms:

a. Pellicle: A mass of organisms is floating on top of the broth.

b. Turbidity: The organisms appear as a general cloudiness throughout the broth .

c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube.

2. Slant tubes: are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface .

3. Stab tubes (deeps): are tubes of hardened agar medium which are inoculated by "stabbing" the inoculum into the agar .

4. Agar plates: are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.

Agar Stab Culture of a Bacterium An Agar Slant Tube

Agar Plate Culture of a Bacterium

Three common forms of agar media.

Inoculation of agar slants and deeps.

End of lecture