bacteriology act8 laboratory | Exercises Bacteriology

MEDIA INOCULATION

Aseptic technique

The media on which you culture desirable microorganisms will readily grow undesirable

contaminants, especially molds and other types of fungus, and bacteria from your skin

and hair. It is therefore essential that you protect your cultures from contamination from

airborne spores and living microorganisms, surface contaminants that may be on your

instruments, and from skin contact.

Bacteria and other contaminants cannot fly. Nearly all forms of contamination are

carried on microscopic dust particles that make their way onto sterile surfaces when they

are carelessly handled. One exception is insect contamination, such as by ants for fruit

flies. Fruit flies are a particular nuisance because they can crawl under the lids of agar

plates and lay eggs.

Never leave a culture dish open, even for a short time when viewing colonies of

organisms, unless you intend to destroy it.

• When it is necessary to open a dish, keep the lid close to the dish, open it only as

far and as long as is necessary to accomplish the procedure, and keep the lid

between your face (and your germs!) and the agar surface.

• For most bacterial cultures you will use a sterile loop or needle to inoculate or to

obtain an inoculum.

• Flame a loop or needle to red-hot just prior to use, burning off any organic material

• Cool the instrument by touching the sterile agar or liquid surface prior to touching

a culture (or else you will kill it)

• Re-sterilize the instrument after performing the procedure, putting down safely

without burning the bench, you, or another student.

• Pass the neck of a culture tube or any container with a culture or sterile contents

through a flame before taking off the cap. Hold the cap with opening down, and

the tube horizontal or nearly so. Convection from the heated neck will prevent

dust from falling into the opening. Flame again before putting the cap back [see

'preparing a bacterial smear' in the staining section]

• Use sterile disposable pipets to remove samples from a broth culture that must be

kept uncontaminated.

• Always be aware of where your hands are, where your face is, and whether or not

your culture is in a position to be contaminated. If you have long hair, make sure

it does not hang into your plate. Hair is full of potential contaminants, and is one

of the principle sources of contaminating microorganisms.

• If you have an open flame, long hair that is not tied back or loose clothing can be

hazardous to your health.

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MEDIA INOCULATION

Aseptic technique The media on which you culture desirable microorganisms will readily grow undesirable contaminants, especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact. Bacteria and other contaminants cannot fly. Nearly all forms of contamination are carried on microscopic dust particles that make their way onto sterile surfaces when they are carelessly handled. One exception is insect contamination, such as by ants for fruit flies. Fruit flies are a particular nuisance because they can crawl under the lids of agar plates and lay eggs. Never leave a culture dish open, even for a short time when viewing colonies of organisms, unless you intend to destroy it.

When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface.
For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum.
Flame a loop or needle to red-hot just prior to use, burning off any organic material
Cool the instrument by touching the sterile agar or liquid surface prior to touching a culture (or else you will kill it)
Re-sterilize the instrument after performing the procedure, putting down safely without burning the bench, you, or another student.
Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking off the cap. Hold the cap with opening down, and the tube horizontal or nearly so. Convection from the heated neck will prevent dust from falling into the opening. Flame again before putting the cap back [see 'preparing a bacterial smear' in the staining section]
Use sterile disposable pipets to remove samples from a broth culture that must be kept uncontaminated.
Always be aware of where your hands are, where your face is, and whether or not your culture is in a position to be contaminated. If you have long hair, make sure it does not hang into your plate. Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms.
If you have an open flame, long hair that is not tied back or loose clothing can be hazardous to your health.

Keep flammables away from the flames, including alcohol used for sterilizing instruments; do not place a heated loop or glass rod into an alcohol dish A contaminated culture can often be rescued, however there is always the risk that you will re-isolate the wrong microorganism. Besides, you don't have that kind of time to waste. Exercise extreme care to keep your cultures pure. Using a sterile cabinet Unlike a fume hood, which is designed to keep airborne substances from escaping into the laboratory environment, a sterile cabinet keeps airborne contaminants from getting into the hood. A simple laminar flow hood protects exposed sterile surfaces that are placed inside. A containment hood does both jobs, keeping airborne particulate matter from going in or out. To use a hood properly, remember these points.
Keep all surfaces clean and dry
Frequently use the UV light to sterilize the interior surfaces; do not stare at the light, which can cause retinal damage
The opening must not exceed the recommended sash height
Surfaces kept to back of the hood are more likely to remain sterile.Keep non-sterile objects closer to the front, sterile objects to the back
Keep the hood fairly uncluttered
Never reach over a sterile surface - you WILL contaminate it; reach around sterile surfaces if necessary
Watch for long hair hanging over sterile surfaces
Use slow, deliberate movements to avoid inadvertant contamination Accumulated waste materials can pose a contamination hazard. A microbiology laboratory can become inundated with old cultures unless a well organized system for disposal of is in place. Even a few people can produce so much contaminated material, that if teams don't take care of their own materials someone will spend at least a week just cleaning up the place. All cultures must be sterilized before disposal or cleaning of lab ware. To make disposal as efficient as possible, please get rid of materials you no longer need as soon as possible. CULTIVATION AND COLONIAL CHARACTERISTICS OF BACTERIA In order to work with microorganisms in the laboratory, it is desirable to obtain them in pure cultures. Pure cultures of bacteria can be obtained by spreading bacteria out and permitting the individual cells to form masses of growth called colonies. One can then pick a sample from the colony and be assured that it contains only one kind of bacteria. Cultivating these bacteria on a separate medium will yield a pure culture.

Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The pour plate technique. Spread Plate Technique The spread plate technique is used for enumerating microorganisms. *Drop 0.1 ml aliquots from serial dilutions onto the surface of an agar plate. Aseptically spread inoculums across the surface using bent glass rod of sterile inoculating loop. By spreading the suspension over the plate, a dilution gradient is established to provide isolated colonies. Incubate and count colonies.

Pour Plate Technique In this method, a sample of bacteria is diluted in several tubes of melted medium such as nutrient agar. After dilution, the melted agar is poured into separate Petri dishes and allowed to harden. Since the bacteria have been diluted in the various tubes, the plates will contain various dilutions of bacteria, and where the bacteria are most diluted, they will form isolated colonies. *Perform serial dilution of sample. Aseptically pipette microorganism dilutions into labeled petri dishes. Add liquefied agar that has been cooled to approximately 44-45 C. Mix well by slightly rotating plate with bacteria and agar mixture. Allow the agar to solidify, trapping the bacteria at separate discrete positions within the medium. Incubate and count colonies. Streak/Stab Method for Agar Tubes Tube media may be in the form of solid agar slants, semisolids or broths. Depending on the type of medium used and the purpose of the inoculation, use an inoculating loop or needle. *For agar slants, place the loop at the base of the tube surface and draw it up the agar surface while moving it from side to side. *For butts, insert the loop into the medium to approximately one fourth of its depth. If testing motility, use needle and stab it in the center of the agar tube to the bottom. Draw the needle out carefully, keeping it straight. Inoculation of Broth Media Broth media are generally used as enrichments, general cultivation and sterility testing. *Aseptically inoculate appropriate broth media with the sample or specimen using sterile pipette, syringes or forceps. Incubate inoculated broth. Examine broth for any signs of growth including turbidity with or without gas bubbles, puff-ball appearance, hemolysis (Blood cultures), pellicle formation and precipitate on the bottom of the tube or bottle.

Name:______________________________ Date Completed: _____________________________ Class:____________ Lab Minutes:______________ Teacher: _______________________________ ACTIVITY No. 6 STREAK METHOD FOR AGAR PLATES Objectives:

To develop skills in bacterial cultivation
To develop skills in streak method using agar plates Materials needed: Prepared NA plates from exercise 9 Inoculating loop Stock culture of a microorganism Alcohol lamp Marker Incubator Gloves Mask Procedure:

While wearing gloves, sterilize an inoculating loop by placing it at an angle over a flame. The loop should turn orange before you remove it from the flame.
Remove the lid from a culture plate containing the desired microorganism.
Cool the inoculating loop by stabbing it into the agar in a spot that does not contain a bacterial colony.
Pick a colony and scrape off a little of the bacteria using the loop. Be sure to close the lid.
Using a new agar plate, lift the lid just enough to insert the loop.
Streak the loop containing the bacteria at the top end of the agar plate moving in a zig-zag horizontal pattern until 1/2 of the plate is covered.
Sterilize the loop again in the flame and cool it at the edge of the agar away from the bacteria in the plate that you just streaked.

Rotate the plate about 45 degrees and spread the bacteria from the end of the first streak into a second area using the same motion in step 6, this time covering ¼ of the plate.
Sterilize the loop again using the procedure in step 7.
Rotate the plate about 60 degrees and spread the bacteria from the end of the second streakinto a new area in the same pattern as procedure 8.
Sterilize the loop again.
Replace the lid and invert the plate. Incubate the plate overnight at 37 degrees Celsius (98.6 degrees Fahrenheit).
You should see bacterial cells growing in streaks and in isolated areas. Tips:
When sterilizing the inoculating loop, make sure that the entire loop turns orange before using on the agar plates. 2. When streaking the agar with the loop, be sure to keep the loop horizontal and only streak the surface of the agar. Draw the growth on your plates Plate 1 Plate 2

bacteriology act8 laboratory, Exercises of Bacteriology

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MEDIA INOCULATION