It is essential that you obey the following rules:
1. Report all accidents immediately to a member of staff or a demonstrator.
2. Wear a lab coat to protect your clothing from contamination.
3. Do not eat, drink or smoke in the laboratory; avoid all hand to mouth operations.
4. Keep your bench tidy to minimise risk of accidents
5. Avoid any procedures that would create aerosols, e.g. violent agitation of liquid cultures, or bubbling of air into liquid cultures from pipettes.
6. Flammable substances must be kept well away from sources of ignition.
7. Broken glass should be placed only in a "Broken Glass" waste bin, not in an ordinary bin. Other sharp items (razor blades, scalpel blades, needles etc.) should be disposed of in the sharps containers, which you will find on top of the shelf units.
8. Plastic ware and other non-reusable debris: put inside the plastic autoclave bags
9. Glassware: loosen any screw caps and place in the container designated for glassware.
10. Wash your hands thoroughly before leaving the laboratory.
Introduction
One of the most important activities that microbiologists undertake is the isolation and identification of disease-causing micro-organisms. Isolation of these organisms is often required from diseased individuals and a wide range of specimens will be encountered in the diagnostic laboratory. These include blood, urine, faeces, tissue, and cerebrospinal fluid. In addition to isolating organisms from diseased individuals it is often necessary to determine if pathogens (or indicator organisms) are present in food, water or environmental samples. Many techniques have been developed for the isolation and identification of microbes. Although techniques such as light microscopy, cell staining, culture on various types of agar, growth in different atmospheres and different temperatures have been in use for well over a century these methods still form. the basis of most microbial identification schemes. These are supplemented by a variety of biochemical tests based on the ability to utilise certain sugars or other compounds as energy sources, production of certain specific enzymes, etc. Many of these schemes have been used to produce miniaturised commercial identification kits with large computer databases allowing quick and easy identification of unknown organisms.
When examining foodstuffs it is often necessary to know not only whether an organism is present but in what numbers it is present. This is related to the potential for spoilage or causing or disease (e.g. is an infectious dose present?). There are many methods for determining the number of cells present in a sample. One of the most useful is the viable count. This allows a determination of the number of cells in a given amount of material (usually expressed as colony forming units (CFU) per gram or per millilitre) that are able to reproduce (and hence form. colonies on an appropriate solid media).
Aim
You will be provided with a food sample weighing 10g. This sample will be analysed for the presence of a variety of indicator organisms and foodborne pathogens.
i. The first step in this process is to homogenise the food sample to release the bacteria into a liquid medium and produce a suspension where the bacteria are evenly distributed.
ii. The next step is to make serial dilutions from this homogenate.
iii. These serial dilutions are plated out onto different types of agars to determine what bacteria are present in the sample and what concentration they are present at. Results will be collected after a few days of incubation.
iv. Finally, further analysis and identification tests will be conducted on isolates after the incubation of the plates is completed.
SESSION 1
Work in groups for this experiment.
Making a food homogenate
Method
1) Pour 90ml of sterile Maximum Recovery Diluent (MRD) into the bag containing your food sample.
2) Place the bag containing the food sample into the stomacher and stomach it for 30sec at 230rpm (your demonstrator will show you how). This will homogenise the food sample and ensure that any bacteria are released into the resulting liquid homogenate.
3) Remove the bag containing the homogenate. Allow the solid debris to settle, then pipette 10ml of the homogenate into a sterile test tube. This homogenate is a 1 in 10 or 10-1 dilution of your food sample. Label it as 10-1.
Serial Dilutions
1) You have been supplied with test tubes with 9ml of MRD pre-dispensed into them. Label each test tube with the following numbers: 10-2, 10-3, 10-4, 10-5, 10-6.
2) Remove 1ml of homogenate you have been provided with and add it to the tube marked 10-2.
3) Mix the contents of the 10-2 tube thoroughly then remove 1ml and transfer to the 10-3 tube.
4) Mix the contents of the 10-3 tube thoroughly then remove 1ml and transfer to the 10-4 tube.
5) Mix the contents of the 10-4 tube thoroughly then remove 1ml and transfer to the 10-5 tube.
6) Mix the contents of the 10-5 tube thoroughly then remove 1ml and transfer to the 10-6 tube.
7) Mix the contents of the 10-6 tube thoroughly.
Plating out serial dilutions:
We will now inoculate different types of agar plates with our serial dilutions to determine different characteristics of our food sample. The different tests we will do are:
1) Aerobic Colony Count (ACC)
This allows us to determine the microbial loading of the food. All organisms present that can grow aerobically and whose nutritional requirements are met by Nutrient Agar will grow. This test is undertaken by the spread plate method.
Method:
1) Label the back of your nutrient agar plates with your initials, the date and the dilution factor (i.e.10-1, 10-2 etc).
2) Mix the contents of the 10-6 tube thoroughly and pipette 0.1ml of the dilution onto the surface of the nutrient agar plate labelled 10-6.
3) Remove a sterile plastic spreader from the packet and then move the spreader backwards and forwards over the surface of the inoculated agar plate. Ensure the liquid is distributed thoroughly and spread evenly over the surface of the agar.
4) Repeat steps 2 &3 with each serial dilution you made beginning with the most dilute (i.e. 10-5 dilution) and working through to the most concentrated (i.e. the undiluted homogenate).
5) Allow the plates to sit on the bench, lid uppermost until all the liquid has been absorbed.
6) Incubate the plates at 37oC for 24 hours.
2) Enumeration of Enterobacteriaceae
This allows us to determine the number of bacteria belonging to the family Enterobacteriaceae (e.g. organisms such as E. coli, Citrobacter, Salmonella, Shigella etc.) that are present in our food sample. We use the selective and differential agar Violet Red Bile Glucose (VRBG) agar to perform. this test and we use the pour plate method.
Method:
1) Label the back of the empty Petri dishes on your bench with your initials, the date and the dilution factor (i.e.10-1, 10-2 etc).
2) Mix the contents of the 10-6 tube thoroughly and pipette 1ml of the dilution into the Petri dish labelled 10-6.
3) Pour 15ml of molten VRBG agar (in bottles in the 45oC water bath) into the Petri dish. Mix the bacterial suspension and agar by gently moving the Petri dish backwards and forwards 4 times, left to right 4 times followed by rotating the Petri dish clockwise 4 times then anticlockwise 4 times.
4) Repeat steps 2 &3 with each serial dilution you made beginning with the most dilute (i.e. 10-5 dilution) and working through to the most concentrated (i.e. the undiluted homogenate).
5) Allow the agar to solidify (this will take 10-15min at room temp) before placing the plates in the tray for incubation. Incubate the plates at 37oC for 24 hours.
3) Isolation & Enumeration of Bacillus cereus
