1. GRAM STAIN
RESULTS
Complete the following table with your results. Compare your results with the expected results table provided by the lecturer.
| Bacterial name | Gram reaction | Shape and arrangement of cells |
| Staphylococcus aures | Purple | -staphylococci |
| Escherichia coli | Light pink | -coccobacillus The arrangement is close together |
| Lactobacillus plantarum | Purple | -bacillus The cells are larger and the colour is darker |
( /3 marks)
QUESTIONS
1. What factors are important in achieving a good Gram stain? List them.
( / 3 marks)
2. What is the difference between a simple and a differential stain?
( / 2 marks)
3. What part of the bacterial cell is most involved with Gram staining and why?
( /2 marks)
In gram staining, the cell wall is the most important part of the bacterial cell. This is due to the fact that the two varieties of gram bacteria, gram positive and gram negative bacteria have different and distinct cell wall constituents.
Class results
| PREPARATION USED | AMOUNT OF GROWTH | |
| BEFORE WASHING | AFTER WASHING | |
| Soap | ++ | +++ |
| Carex | + | +++ |
| Hydrex | +++ | + |
| Purell | ++ | + |
| Lab soap | +++ | ++ |
| Carex | – | ++ |
1. How did the microbial flora before washing vary between people and why might it vary?
( /3 marks)
2. Which preparation was the most effective? Which was least effective? Why are some preparations better than others?
( /4 marks)
VIABLE COUNTING OF MICROORGANISMS
RESULTS
Carry out the calculations of the following results, ensure that you follow counting guidelines and that you express your results in standard form:
| Counting Method | Dilution | Number of colonies | Count/ml (show your calculations) |
| Pour plate | 10-6 | 47 | CFU/ml = |
| 10-7 | 75 | ||
| 10-8 | 1 | ||
| Spread plate | 10-5 | NA (TMTC) | |
| 10-6 | 191 | ||
| 10-7 | 61.5 |
( / 4 marks)
QUESTIONS
1. When performing serial dilutions for viable counts there are a number of possibilities for error which may lead either to an underestimate or an overestimate of the actual number of bacteria present. Study the list of possible errors below and indicate whether the error will lead to U (underestimate), O (overestimate) or NE (no effect)
| POSSIBLE ERROR | UNDERESTIMATE OVERESTIMATE NO EFFECT |
| High carryover of cells from low ® high dilution | |
| Volume of diluent in tubes lower than intended | |
| Volume transferred higher than intended | |
| Low level contamination of low dilution tube | |
| Loss of viability during dilution | |
| Plate medium does not support good growth of organism |
0.5 marks per right answer
( / 3 marks)
2.
your results and compare the methods used.
Remember that you measured the number of bacteria from the same culture – were the counts the same? (Max 300 words)
( / 5 marks)
MICROBIOLOGICAL EXAMINATION OF FROZEN PEAS
RESULTS
1. BACTERIAL COUNTS (PCA PLATES)
| DILUTION | NUMBER OF COLONIES | Number of orgs per g of food | ||
| Plate 1 | Plate 2 | Average | ||
| 10-5 | ||||
| 10-6 | ||||
| 10-7 |
2. ENTEROBACTERIA COUNTS (VRBLA PLATES)
| DILUTION | NUMBER OF COLONIES | Number of Enterobacteria per g of food | ||
| Plate 1 | Plate 2 | Average | ||
| 10-1 | ||||
| 10-2 |
3. YEAST AND MOULD COUNTS (RBCA PLATES)
| DILN | NUMBER OF COLONIES | Number of fungi per g of food | ||
| Plate 1 | Plate 2 | Mean | ||
| 10-2 | ||||
| 10-3 | ||||
| 10-4 |
( / 6 marks)
Class results
| Sample | Total viable counts | Coliforms | Fungi |
| A | 2.2 x 102 | 0 | 3 x 102 |
| 2.9 x 103 | 0 | 1.1 x 102 | |
| 9.5 x 102 | 0 | 1.6 x 103 | |
| B | 3.25 x 104 | 4.0 x 101 | 3.05 x 102 |
| 6.4 x 104 | 4.7 x 103 | 7.8 x 103 | |
| 9.6 x 105 | 3.0 x 101 | 6.1 x 102 | |
| 1.63 x 104 | 3.25 x 102 | 3.95 x 102 | |
| C | 3.5 x 105 | 2.05 x 103 | 3.5 x 102 |
| 2.3 x 106 | 6.45 x 102 | 1.8 x 103 | |
| 4.75 x 106 | 2.5 x 101 | 8.0 x 102 | |
| 1.4 x 106 | 1.32 x 103 | 7.2 x 103 | |
| D | 4.2 x 109 | 2.68 x 104 | 4.3 x 107 |
| 2.47 x 109 | 7.04 x 104 | 6.35 x 106 | |
| 3.4 x 109 | 1.89 x 105 | 1.81 x 106 | |
| 5.65 x 109 | 2.54 x 105 | 2.02 x 107 | |
| 1.51 x 109 | 9.9 x 104 | 5.05 x 107 | |
| 1.3 x 109 | 6.4 x 104 | 1.66 x 106 | |
| 4.0 x 109 | 8.2 x 104 | 9.3 x 106 |
DISCUSSION / QUESTIONS
1. Discuss the differences in the microbial populations of samples A to D in terms of numbers and types of microorganisms. (Max 500 words)
( / 5 marks)
What are the likely sources of microorganisms causing spoilage of the peas? List two.
( / 2 marks).
MICROBIOLOGICAL EXAMINATION OF READY TO EAT FOODS
1. Total viable count (Blood Agar Base) results
| DILUTION | NUMBER OF COLONIES | Number of orgs per g of food | ||
| Plate 1 | Plate 2 | Average | ||
| 10-6 | ||||
| 10-5 | 102 | 207 | 154.5 | |
| 10-4 | 77 | TMTC | ||
| 10-3 | 199 | 52 | 125.5 | |
| 10-2 | TMTC | 157 | ||
| 10-1 |
2. Enterobacteria count (VRBLA)
| DILUTION | NUMBER OF COLONIES | Number of Enterobacteria per g of food | ||
| Plate 1 | Plate 2 | Average | ||
| 10-1 | ||||
| 10-2 | ||||
| 10-3 |
3. Listeria count (MMA)
| DILN | NUMBER OF COLONIES | Number of Listeria per g of food | ||
| Plate 1 | Plate 2 | Average | ||
| 10-1 | 59 | 74 | 66.5 | = 2660 |
( / 6 marks)
Class results
| Food sample | Aerobic colony count (cfu/g) | Enterobacteria (cfu/g) | Listeria (cfu/g) | Quality designation (see HPA guidelines) |
| A | 1.28 x 104 1.65 x 103 9.8 x 106 7.4 x 103 7.4 x 103 | 2.71 x 104 4.0 x 102 5.2 x 102 3.25 x 102 6.5 x 102 | 2.4 x 102 6.8 x 102 2.86 x 103 5.8 x 102 1.8 x 102 | |
| B | 1.89 x 104 3.4 x 104 6.65 x 104 | 6.1 x 102 3.25 x 102 2.66 x 103 | 3.88 x 103 5.8 x 102 0 | |
| C | 1.59 x 104 1.10 x 105 7.0 x 104 2.3 x 105 5.5 x 104 | 1.58 x 103 6.2 x 103 4.45 x 102 3.62 x 103 4.9 x 102 | 1.64 x 103 3.72 x 103 1.06 x 103 1.66 x 103 5.2 x 102 | |
| D | 1.66 x 107 | TMTC | 0 | |
| E | 5.4 x 105 2.04 x 106 | 4.95 x 102 3.65 x 104 | 4.0 x 101 2.4 x 103 |
( /6 marks)
HPA Guidelines for the microbiological quality of sandwiches
| Criterion | Microbiological quality (cfu/g unless stated) | |||
| Satisfactory | Acceptable | Unsatisfactory | Unacceptable / potentially hazardous | |
| Aerobic colony count | <106 | 106-<107 | ≥107 | N/A |
| Enterobacteria E. coli | <100 <20 | 100-<104 20-<100 | ≥104 ≥100 | N/A N/A |
| Listeria L. monocytogenes | <20 <20 | 20-<100 20-<100 | ≥100 N/A | N/A ≥100 |
| Salmonella | Not detected in 25g | Detected in 25g |
From: Gilbert, R.J., de Louvois, J., Donovan, T., Little, C., Nye, K., Riberio, C.D., Richards, J., Roberts, D. and Bolton, F.J. Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. Comm. Dis. Pub. Health 2000; 3: 163-167.
Grades of microbiological quality
The terms used to express the microbiological quality of the ready-to-eat foods are:
Satisfactory: test results indicating good microbiological quality
Acceptable: an index reflecting a borderline limit of microbiological quality
Unsatisfactory: test results indicating that further sampling may be necessary and that environmental health officers may wish to undertake a further inspection of the premises concerned to determine whether hygiene practices for food production or
handling are adequate or not.
Unacceptable/potentially hazardous: test results indicating that urgent attention is needed to locate the source of the problem; a detailed risk assessment is recommended. Such results may also form a basis for prosecution by environmental health departments, especially if they occur in more than one sample. Food examiners will wish to draw on their own experience and expertise in determining the advice and comments they wish to give and they will be required to do this if invited to give an expert opinion during legal proceedings.
Questions:
1. Discuss the class results. Do all the products tested comply with their specified microbiological limits? (Max 500 words)
( / 5 marks)
2. What are the main sources of microbial contamination in the production of sandwiches? List 3 possible sources and explain how they would affect microbial counts. (Max 300 words)
( / 3 marks)
3. Outline the approaches used in the food industry during manufacture to ensure that foods of the type tested are microbiologically safe. The blanket mention of HACCP will not be allocated any marks. (Max 300 words)
( / 3 marks)
Fungal Spoilage of Fruit – RESULTS
Observe your own samples for the extent of fungal infection – e.g. softening, discoloration, leakage of juice and appearance of fungal mycelium away from inoculum site.
1. Oranges / Penicillium
| TREATMENT | ASSESSMENT OF FUNGAL GROWTH / FRUIT SPOILAGE |
| NO TREATMENT | No leakage No fungal mycelium No discolouration Not soft its rather hard texture |
| WAX REMOVED | No leakage No fungal mycelium No discolouration Slightly soft texture |
| SURFACE ABRADED | No leakage A few fungal mycelium Slight discolouration |
| SKIN CUT | No leakage Present of fungal mycelium Slight discoloration |
| BLOCK CUT OUT | Slight leakage Fungal mycelium present Discolouration present Soft texture |
2. Apples / Rhizopus
| TREATMENT | ASSESSMENT OF FUNGAL GROWTH / FRUIT SPOILAGE |
| NO TREATMENT | Hard texture No discolouration No leakage No fungal mycelium |
| WAX REMOVED | Hard texture No discolouration No leakage No fungal mycelium |
| SURFACE ABRADED | Soft texture Discolouration present Leakage from the apple Fungal mycelium present |
| SKIN CUT | Hard texture No leakage No discolouration Small fungal mycelium present |
| BLOCK CUT OUT | Hard texture no leakage Discolouration present Soft texture fungal mycelium |
Questions (remember in your answers to refer and give details of your observations):
1. How does the amount of treatment affect the level of spoilage for each of the fruit? (Max 500 words)
( /5 marks)
2. In what way do the two different types of fruit spoil? What characteristics of each type of fruit may lead to these differences? (Max 400 words)
( /4 marks)
Marks grid
| PRACTICAL | MARK |
| Gram stain | /10 |
| Handwashing | /7 |
| Viable counts | /12 |
| Microbial examination of frozen peas | /13 |
| Microbial examination of ready to eat food | /17 |
| Fungal spoilage of fruit | /9 |
OVERALL MARK: /68 à %
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