代做Isolation & Enumeration Of Microbes From Food 2023-24调试Haskell程序

Isolation & Enumeration Of Microbes From Food

Practical Guide 2023-24

School of Chemical Engineering

Safety Instructions

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

Using the agar PEMBA (Polymyxin pyruvate egg yolk mannitol bromothymol blue agar) we can test for the presence and level of B. cereus (spores and/or vegetative cells) in foodstuffs. PEMBA is a selective and differential agar and the test is performed by the spread plate method.

Method

Repeat the method described in section (1) ‘Aerobic Colony Count’ using PEMBA media.

4) Isolation & Enumeration of Clostridium perfringens

Using the agar TSC (Tryptose sulphite cycloserine agar) we can test for the presence and level of C. perfringens (spores and/or vegetative cells) in foodstuffs. TSC is a selective and differential agar and the test is performed by the pour plate method.

Method:

Repeat the method described in section (2) ‘Enumeration of Enterobacteriaceae’ using TSC media.

SESSION 2

Check each set of plates that you inoculated for growth. By examining the number of colonies and their appearance on each type of agar you will be able to determine the concentration of each organism or group of organisms in your food sample.

1. ACC: Choose the nutrient agar plate from your dilution series that has between 30 and 300 colonies on it. Count all the colonies and determine the ACC per gram of food using the following formula.

2. Enterobacteriaceae: Choose the VRBG agar plate from your dilution series that has between 30 and 300 colonies on it. Colonies of organisms belonging to the enterobacteriaceae should have the following appearance: purple red in colour, 0.5mm in size (or larger), may be surrounded by a red zone of precipitated bile. Count all the colonies on your chosen plate that conform. to the description given above. Determine the enterobacteriaceae count using the following formula.

3. B. cereus: Choose the PEMBA plate from your dilution series that has between 30 and 300 colonies on it. B. cereus colonies should have the following appearance: large (approximately 5 mm diameter), crenated colonies, a distinctive turquoise to peacock blue colour and usually surrounded by a zone of egg yolk precipitate of the same colour. Count all the colonies on your chosen plate that conform. to the description given above. Determine the B. cereus count using the following formula.

4. C. perfringens: Choose the TSC plate from your dilution series that has between 30 and 300 colonies on it. Count only black colonies. Determine the C. perfringens count using the following formula.

Formula: cfu/g = number of colonies x dilution factor x dilution factor related to the amount plated (or cfu/ml if the original sample was liquid)

a. number of colonies: The number of colonies on the plate (using plates with 30-300 colonies is considered statistically valid).

b. dilution factor: The dilution factor refers to the dilution used for counting the colonies. For example, if the 10-4 plate was counted then the dilution is 104.

c. dilution factor related to the amount plated: for example if 0.1 ml was plated on the plate then the dilution factor should be 10 in order the final result to refer to 1 ml (or 1 g). This is because 0.1 ml x 10 = 1 ml.

Confirmatory testing:

Isolates on selective and differential plates can only be regarded as presumptive isolates of the organism you are looking for. To confirm that they are what you suspect them to be some confirmatory tests need to be undertaken.

Enterobacteriaceae: Isolates from VRBG plates have been sub-cultured for you onto Nutrient agar plates. Use these subcultures to perform. an oxidase test, catalase test and Gram staining as follows:

Oxidase test: Remove an oxidase test stick from the container. One end of the stick has a pale pink band around it. Touch this on a single isolated colony on a nutrient agar plate. After 30 seconds observe the colour – if the pink band changes to a bright purple colour the organism is oxidase positive. If no colour change is observed it is oxidase negative. Dispose of the oxidase test sticks in the contaminated plastics disposal bag.

Catalase test: in a sterile petri dish pipette a few drops of regent (hydrogen peroxide). Use a sterile tip to pick up and mix your cells with the reagent. Creation of bubbles indicates a positive reaction (eye protection is required).

Gram Stain: Enterobacteriaceae are small Gram- negative rods. Pick a colony and Gram stain it according to the method listed in appendix. View this under 100x magnification on the microscopes provided.

Colony and cell characteristics: Record the colony and cell characteristics (see appendix).

B. cereus: B. cereus cells may contain sub-terminal spores that do not swell the sporangium. Pick a colony and Gram stain it according to the method listed in appendix. View this under 100x magnification on the microscopes.

Colony and cell characteristics: Record the colony and cell characteristics (see appendix).

C. perfringens: Isolates from TSC plates have been subcultured for you onto Blood agar plates. One was incubated aerobically and the other anaerobically. Examine these for growth. Organisms that only grow on the anaerobic plate and do not have a diffuse spreading colony type (which indicates motility) are presumptive C. perfringens.

Perform. a Gram stain on one of these colonies. Gram Stain: C. perfringens cells that may contain sub-terminal/central spores. Pick a colony and Gram stain it according to the method listed in appendix. View this under 100x magnification on the microscopes provided.

Colony and cell characteristics: Record the colony and cell characteristics (see appendix). ACC: Isolates have been subcultured for you onto Nutrient agar plates. Use these subcultures to perform. an oxidase test, catalase test and Gram staining (see appendix).

Colony and cell characteristics: Record the colony and cell characteristics (see appendix).

ASSESSMENT- LABORATORY REPORT

Approximately 1000 – 1500 words (excluding references) using the following format:

Introduction

Review and discuss the relevance and aims of the experiment. Specifically, review the literature regarding microbes related to your selected food sample and present the aim of the experiment (analyses) within this context.

Methodology

- Brief overview of the methods focusing on their key principals and their relevance/purpose in this practical (no details about the method procedures as these have been given in the course document).

- Which of the media you are using are selective, non-selective, differential and what ingredients make them selective?

Results

Record and present your data in a table:

- Colony characteristics (see appendix)

- Cell characteristics (see appendix)

- Confirmatory tests: oxidase/catalase tests, Gram staining etc.

- cfu/g of food for the particular microorganisms (including your calculation showing correct formulas

Discussion and conclusions

1. Presumptive identification of isolate from ACC: Consider your results in order to identify your unknown (suggest possible genus, species etc.). Also, take into account the type of food that it was isolated from. Support your conclusions with relevant literature and resources.

Note that your unknown came from a non-selective medium, therefore possible identities are not limited to the microbial genera and species of the reference strains that were provided during this practical.

2. Discuss the significance of microorganisms in food samples with reference to your findings (detection and quantification of the microbial groups that were targeted in this practical).

3. Consider the factors involved in isolation, identification and quantification of micro-organisms from food:

a. What are the limitations of the methods? Make suggestions for further study (focus on alternative approaches and methodologies that could be used for achieving and/or expanding the aim of this investigation.

b. Could some bacteria grow on the pour plate and not be seen using the spread plate technique?

Explain.