TITLE: TRANSFER & ISOLATION TECHNIQUES OF MICROBIOLOGY
OBJECTIVE:
1. To transfer bacteria aseptically between tubes of growth media
2. Aseptically perform a streak plate resulting in isolated colonies.
MATERIAL:
Mixed culture (Broth, agar slant, agar plate)
Bunsen Burner
Inoculating Loop
Sterile broth, agar slant & agar plate
Alcohol 70 %
PROCEDURE:
Inoculation of a broth culture (broth to broth transfer)
1. The sterile nutrient broth is labeled with the source of culture and initials.
2. The loop is sterilized.
3. Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.
4. The loop is inserted into sterile broth tube and gently swirl. The loop is sterilized.
5. The broth is incubate at 37’C for 24-48 hours.
6. The broth is observed for turbidity. The result is recorded in table at the end of procedure section.
Inoculating on agar slant (Broth to slant transfer)
1. The sterile nutrient agar slant is labeled with source of culture and initials
2. The loop is sterilized.
3. Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.
4. The loop is inserted into the sterile agar slant tube and starting at the base of the slant, the loop is draw up the slant. The agar not penetrate. The loop is sterilize.
5. The slant is incubated at 37’C for 24-48 hours.
6. The slant for growth is observed. The result is recorded in the table at end of the procedure section.
Streak plate ( Plant culture to Plant transfer)
1. The sterile nutrient agar slant is labeled with source of culture and initials
2. The loop is sterilize.
3. Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed
4. The agar plate is lifted from the lid and streak about the half of plate. The loop is be in parallel to the agar surface to prevent digging into gouging the agar.
5. The plate of lid is returned. The loop is sterilized. The agar plate is lifted and make one streak into the inoculated portion of the plate. By streaking, the remaining one-fourth of the uninoculated plated is finished.
6. The plate of lid is returned. The loop is sterilized. The agar plate is lifted and make one streak into the second inoculated portion of the plate. By streaking, the remaining one-fourth of the uninoculated plated is finished. The loop is sterilize.
7. The plate is placed in a 37’C incubator for 24-48 hours. The growth is observed and result is recorded in table provided at the end of the procedure section.
Inoculating on agar slant (slant to slant transfer)
1. The sterile nutrient agar slant is labeled with source of culture and initials
2. The loop is sterilized.
3. Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.
4. The loop is inserted into the sterile agar slant tube and starting at the base of the slant, the loop is draw up the slant. The agar not penetrate. The loop is sterilize.
5. The slant is incubated at 37’C for 24-48 hours.
6. The slant for growth is observed. The result is recorded in the table at end of the procedure section.
DISCUSSION:
A mixed culture contains two or more bacterial species that are known and can be easily separated based on cultural or biochemical characteristics. Culturing techniques provide a means for maintaining adequate nutrition for the organisms so they can continue to survive. As organisms grow in a culture they consume theavailable nutrients and periodically need to be transferred to fresh media to continue to grow. Certain culturing techniques not only provide the organisms with a fresh supply of nutrients but also allow for the separation of bacterial cells to obtain isolated colonies. These culturing procedures are known as isolation techniques.
Streaking is a technique used in microbiology to isolate a pure strain from a single species of microorganism, often bacteria. A microbiological culture can be grown so that the organism can be identified, studied, or tested. A sterile cotton swab or inoculation loop is sterilized and dipped in a broth or patient specimen containing many species of bacteria.
There is some precaution we must take attention, in the streaking procedure, a sterile loop or swab is used to obtain a microbial culture. The inoculating instrument is then streaked lightly over an agar surface. On the initial section of the streak, many microorganisms are deposited resulting in confluent growth, which is growth over the entire surface of the streaked area. However, because the loop is sterilized between streaking different sections, or zones, fewer and fewer microorganisms are deposited as the streaking progresses. Finally, only an occasional microorganism is deposited, because the streaking process dilutes out the sample that was placed in the initial section.
To obtain good results with this technique, the agar surface should be smooth, moist, and free of contamination. However, excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media, can collect on the inside of the lid and sides of the plate. If this water drops down onto the agar surface, spreading and merging of colonies can occur. This is why one should invert the plates after streaking them and when they are incubated.
CONCLUSION:
Based on the result above, student can transfer bacteria aseptically between tubes of growth media and aseptically perform a streak plate resulting in isolated colonies. Thus, the objective achieved.
QUESTION:
1. How can you tell growth has occurred in a broth culture?
It becomes turbid, the odor may change and if there is an indicator (that is a pH indicator) the color may change.
2. What is the purpose of agar?
Agar is used to solidify microbiological media. Having a solid media allows microbiologists to see colonies of bacteria this eases the identification of a single microbe in a mixed population it also allows the selection of variants in a population, it perhaps allows identification from colonial morphology and enumeration of bacteria. Solid media in lawn cultures (spread plates) are used to calculate zones of diffusion/inhibition. Agar is used rather than other substances as relatively few microbes can manufacture the digestive enzymes required to break it down, so the solid media does not become liquid.
3. At what temperature does agar liquefy and solidify?
0.7% agar will solidify at around 30 deg C but it will stay solid after that at much higher temperatures and liquefy at low than 30’C.
4. Why is liquefied agar cooled to 60’ C before adding organisms?
To improve the motility of medium agar
5. What is primary purpose of the streak plate?
Isolate a single cell by dilution
REFFERENCES:
fda.gov/Food/ScienceResearch/LaboratoryMethods/.../ucm073598.htm
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