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Microbiological risk analysis is done mainly to determine the risk from food which is been consumed, and to minimise it. Also it has been done to ensure the quality of food which is being consumed, to determine total number or people who are ill in the population, to estimate the rate of illness, pathogen population. (red big book)
National government departments as well as international bodies like European scommunity legislation (EC), World Health Organisation (WHO), the International Commission on Microbiological Specifications for Food (ICMSF) and the Codex Alimentarius Commission are working continuously since almost 10 years for food safety in relation to human health and applying these processes of risk assessment to fix the appropriate policies for cosumer protection. (fmb n lab practs)
Food and variety of food products are important aspects for international trading. And loss of export market may have an earnest economic effect on countries which produces these products. That's the reason it is very essential to ensure that when any one country rejects imported food on ground s that it is not good to consume. (Pink)
There are three important parts Risk assessment, risk management and risk communication which falls under an overall system which is known as risk analysis. This has to provide the qualitative and quantitative health effects resulting from the expose to food borne hazards which is called as Risk estimate.
There are four different steps in risk assessment they are as follows:-
Hazard identification:-In this stage mostly chemical, physical and biological identification of hazards takes place. More concern is given towards micro-organisms and their toxins
Exposure assessment:-It is more concern with the quantitative and qualitative evaluation of intake of hazardous agent.
Hazard Characherization:- In this part quantitative and qualitative evaluation of effects associated with specific hazard that is dose responce is of more concern.
Risk characterisation:-This part includes the estimation of results from above three stages.
Risk management: - It mainly deals with the risk assessors, to decide about different measures to reduce the risk .They also deals with the cost and benefits of critical points and implementation.
Risk communication: - In risk communication part effective interaction and communication between risk assessors, risk managers consumers and different interested parties takes place. This is part is one of the important part in whole risk analysis.
Variety of goals made to minimise the risk of food born diseases from different micro organisms can be used by food industries. (pink)
The microbiological risk assessment has been discussed here is about ready to eat meals. The main reason behind it is, In today's world ready meals have become more popular compared to traditional home cooked meals (Nissen et al., 2002).There are several reasons behind it as in time, convenience ,easy to prepare etc. Due to these reasons Ready to eat meals are also used in hospitals and old welfare services. i.e served to immune compromised patients. To control microbiological risk associated with each step of the processing of ready to eat meals till the consumer consumes it risk analysis has to be done . If Ready to eat meal is not kept in proper temperature and proper storage conditions as instructed on package then the chances of microbial growth and is more, which may be hazardous to health. So each ready to eat meal should under go hazard identification, exposure assessment, hazard characterisation and risk assessment followed by risk management, risk communication. Mostly hazard can be exposed from three types of damage,physical damage, chemical damage and biological damage.
Prevalence of B.cereus and St.aureus
There are six different type of species in the bacillus cereus group they are as follows:-B.anthracis, B.cereus, B. mycoids, B.psuedomycoids, B. Thuringiensis, B.weihenstephanesis. Among these species B. Cereus specie is the one main specie which causes food born disease after consumption of contaminated food.This spicie spoils the food quality and becomes hazardous to health.It is gram negative .(red book 445) Dose responce and toxicity of B.cereus is explain as follows:-
If the dose intake goes beyond 105 cells/g then there are 2 types of syndroms takes place
Emetic syndrome:- Type of oxin produced by basillus in this syndrom is cyclic peptude which is stable 126Â°C, 90 min within pH 2-11.Icubation period goes from 0.5-6 hours.Due to this type of toxin person suffers from Abdominal pain, watery diarrhoea, nausea. This may remains from 6-24 hours.this type of syndrom takes place after injestion of Fried and cooked rice, pasta, noodles and potatoes in which bacillus produces toxins.
Diarrhoeal syndrome: Type of oxin produced by basillus in this syndrom Three protein subunits L1, L2, B( Hemolysin BL,Nonhemolytic enterotoxin-Nhe, Cytotoxin K-CytK) which is stable below 56 Â°C, as it gets Inactivated at 56 Â°C for 30 minutes.Icubation period goes from 8-24 hours .Due to this type of toxin person suffers Nausea, vomiting and malaise, water diarrhoea . This may remains from 12-24 hours.this type of syndrom takes place after injestion of Meat, soups, vegetables, fish, puddings, sauces, milk & dairy products in which bacillus produces toxins.(red book pg no 448 and 450 refe-450 table)
In entire life cycle of bacteria sporulation is a resting stage.Two main genera for spore formation are bacillus and clostridium.we are looking for spore formation in bacillus.
Bacteria produce a different type of dormant structures in which he vegetative cell gets converted in to dormant stucture which is called as spoogenesis.(link as reference)
Bacillus species can sporulate only under aerobic conditions.It produces endospores.the procedure of sporulation is consist of 7 to 8 stages which takes place in liquid medium under suitable conditions.During sporulation process at different stages different proteins gets synthesised.
St.aureus is first described in 1879.this bacteria generally occurs in different forms and position like in pairs ,short chains,bunced grap like clusture.it is gram negative bacteria which gives red rod like structure when seen under microscope after performing gram staining. St.aureus is present everywhere as in dust,food,food equipments, milk water, environmental surfaces,humans and animals.Infact Humans and animals are the primary carriers of this bacteria. The main cause of gastroenteritis is injection of food containing toxin produced by st.aureus .This bacteria produces numerous extracellular products which causes illness.It produces wide range of virulence factors and pathogenicity like staphylokinase,hyaluronidases,phosphatase,coagulase and hemolysis.( http://books.google.com/books?id=QWX8wuidSOYC&pg=PT200&dq=information+about+st.aureus&cd=2#v=onepage&q=information%20about%20st.aureus&f=false )St.Aureus produces variety of toxins like:-
Cytotoxins (Alpha, beta, gama, delta,p-v leukocidine):-which are Toxic for many cells including leukocides, erythrocytes, macrophages, platletes and fibroblasts, and causes skin infections
Exfoliative toxin (ETA,ETB):- The toxins have esterase and protease activity which always target a protein .protein is involve in maintaining the integrity of the epidermis. So it causes scalded skin syndrome in neonates
Enterotoxins (A-E, G-I):- It releases the cytokines by Stimulating proliferation of T-cells.it causes high temperature, vomiting, diarrhea, excessive tiredness, aching muscles, confusion, feeling light-headed or dizzy
Same effects can be seen in toxic shock syndrome.( Source: modification of David, M. Rollins lecture, University of Maryland, USA 2006)
Materials and methods
Determination of water activity and pH value of given sample.
The information regarding food sample weight, its nutrient contents, nutrient information were noted down. And did following tests:-
Water activity, aW, is a physical property of food which is more concern with the microbiological safety of food
. "From the physicist's point of view, water activity is defined in terms of
Thermodynamic concepts such as the chemical potential and is related to the osmotic. The aW of a food or solution is the ratio of the water vapor pressure of the food or
Solution (p) to that of pure water (po) at the same temperature: -
aW = p/po
Pressure of an aqueous solution." (Paul Gibbs1 and Vassilis Gekas2
1 Leatherhead Food Research Association. Randalls Road, Leatherhead, Surrey, UK
2 University of Hania, Crete, and Greece).Moisture content of the given food sample were done by keeping the food sample in a white sample dish inside the drawer which is pushed gently into the instrument called Aqualab CX2 .it is generally done to measure the moisture content in the given sample. It is measured as equilibrium relative humidity using an instrument. This instrument works on the Dew-Point principle.(clg booklet).
PH and Temperature:-
The pH Value is used to measure the relative acidity or alkalinity of the ash residue of given food sample. The ash which remains after food sample has been dehydrated can be alkaline, acidic or neutral depending largely upon the mineral content in it. Meat and meat products except the fermented products are a low acid food which is >4.5.(http://books.google.com/books?id=Ac4D3_GHByEC&pg=PA233&dq=ph+of+foods&cd=3#v=onepage&q=ph%20of%20foods&f=false).Most microorganisms grow best with the ph between 6.6-7.5.(Yellow book)
The Ñ€Ð of 10gm of the food sample was measured. To obtain the accurate Ñ€Ð value 10 gm of the lasagne was suspended in 90ml of distilled water. The food added to the distilled water was macerated for 30 seconds in a stomacher. And the Ñ€Ð was measured electronically using a pH meter equipped with a glass electrode.
Enumeration of potential pathogens:
Enumeration of potential pathogens is very important in order to see the concentrate recourses of hazard which are produced .which require hazard identification. This was done by using selective and reliable techniques. As said selective media was used is to control the growth of other unwanted micro flora.
For this the medium was used which is as follows:-
Nutrient agar:-Nutrient agar is important medium to see the overall microbial growth of the product. Nutrient agar contents the nutrients like beef extract, peptone and agar.
Polymixin-egg yolk-mannitol-bromothymol blue agar (PEMBA or B.cereus agar):- This media is selective especially for B.cereus. Even small amount of B.cereus in the food can be recovered by this media.
Violet red blue glucose agar:-
Baird parker agar:- It is a selective medium for the isolation of staphylococci from given food samples. Glycine, lithium and tellurite supports the growth of Staphylococcus aureus and suppress other bacteria in foods .( Department of Health NHS Executive: The Caldicott Committee. Report on the review of patientidentifiable
information. London. December 1997.)
pre-prepared suspension of 10g of the starch moiety of Lasagne were given, which was homogenised with 90ml diluent, i.e. the 10-1 dilution.
From that solution i.e from10-1 to 10-4 dilutions were made.
Dilutions of 100Âµl were then spread plated on to 4 plates of nutrient agar, PEMBA, VRBG and BPA each.
The nutrient agar plates were then incubated at 25°C for 48 hrs and the PEMBA, VRBG and BPA plates were incubated at 37°C for 24 hrs.
Since our class was in next week we were given freshly prepared (spreaded) agar plates.
We did colony count by counting each separate colony.and calculated the number or micro-organisms grown on the agar plates.
Preliminary confirmation of identity:
Gram staining:- Gram-staining is the most useful and commonly used method in microbiological world.This technique gives valuable information.
There are many microorganisms which are translucent in nature. Due to this they are invisible when conventional light microscopy is used .here staining technique plays an important role. With the help of staining technique those translucent cells becomes visible under microscope.
As smear was prepared from each agar plate containing colonies and then the Gram stain technique was performed.( http://en.wikipedia.org/wiki/Staining)
Catalase Test:- Almost all aerobic bacterias produce certain type of enzyme that is Catalase.which further break downs the toxic peroxides.If if dosent happen then it gets accumulated and protects itself from poisoning.This is where catalase test plays an important role. The presence of catalase enzyme was tested by using a droplet of hydrogen peroxide on the colonies formed (black book pg no-253).The main principle behind this test is to convert hydrogen peroxide to water and oxygen.(rest para write in discussion)
Oxidase test:The main principle behind doing this test is to test the oxidation of colourless reagent tetramethyl p-phenylene diamine hydrochloride ,to get purple colour.(black book pg-254). The oxidase test was thus performed on the colonies formed.
Preparation of isolates of presumptive Bacillus and St. aureus:
Pure culture of B.cereus and St.aureus were made for the future practicles and tests. The slopes prepared were maintained by preparing agar slopes .these slopes were made to prevent drying of culture. These slopes then incubated at 25â° c for 48 hours till the microorganisms are grown. After the growth of the micro organisms it was refrigerated for further use.
Phenotypic identification :-
API Biomerieux systems
This system is the best one to obtain a large quantity of data about the phenotypic characteristics of micro-organisms.API method gives a rapid and very convenient result of identifying the enterobacteriaceae. (http://books.google.com/books?id=tvtJabldoKgC&pg=PA3284&dq=Latex+agglutination+test&cd=2#v=onepage&q=Latex%20agglutination%20test&f=false)
There are different systems for identification of different micro-organisms:-
API 20 E for identification of Enterobacteriaceae (gram negative,oxidase negative)
API 50CHB for identification of bacillus spp.
API 50 CHB can be use for both the genera, but the suspending medium is different.
Each well on the card contained a different substrate dried on to the base of the well. The liquid suspension of the sample was added to the well. And was incubated at 37°C for 24 hrs. The results were read after 24 hrs of incubation for 20 E strips and after 48 hrs for 50CHB.
After taking the readings from the strip API 50CHB , all the readins were entered in the API software to found out the isolates of bacillus spp.
Determination of virulence factors:
i) Detection of Bacillus cereus enterotoxin
Reagents and equipment used for this test is as follows:
TD 951 sensitised latex
TD 952 control latex
TD 954 diluent
Pipette set to deliver 25Âµl, yellow tips
1 ml of 18-24 hrs old brain heart infusion was taken which was subcultured. This was provided by university.
That solution was put in the eppendorf tube and kept for centrifugation for 10 min.
From that tube 20 Âµl of supernatant were transferred in to another eppendorff tube.
Then 25 Âµl of diluents were added in to 2 to 8 numbered wells in both the rows A & B.
25 Âµl of sample (supernatant) were added in well number 1 and 2 in both the rows A & B.
Doubling dilution method was performed by changing the pipette each time.
Then 25 Âµl of sensitised latex (TD 951) was added in to all the wells 1 to 8 in row A.
The control latex (TD 952) was added in all the well 1 to 8 in row in row B.
Then the sample putting lid on it was kept for 24 hours undisturbed.
The result was taken on the next day.
used Pasteur pipettes, etc, on the bench; were directly put in the
ii) To see the virulence factor of haemolysis a well isolated colony from the nutrient agar was streaked on to horse blood agar and was incubated at 37°C for 24 hrs
iii) To see the effect of microorganisms as proteolysis, lipolysis and lecithinase production, a well isolated colony from the nutrient agar was strekplated on to egg yolk agar and was incubated at 37°C for 24 hrs.
Predictive microbiology means "Study of the effects on microbial growth of single controlling factors such as
temperature, pH or water activity, resulted in acceptance that particular microbes of
concern would not grow below certain temperatures, or below a certain pH value or
water activity." (Predictive Microbiology - Quantitative Microbial Ecology
Culture - March 2004
Dr J. Baranyi1 and Dr T.A. Roberts2
1 Biomathematician, Institute of Food Research, Norwich, UK
2 Food Safety Consultant, 59 Edenham Crescent, Reading, RG1 6HU, UK
(formerly Head of Microbiology, Institute of Food Research))
This involvs the computer based program , in which all the data i.e pH, water ctivity, temperature related to given food sample can be enter and we get the results and predictions of different conditions suited for microbial growth.
Oxidase staphylase test
Latex agglutination test is best known for its rapid and easy determination of presence of the coagulates of S.aurius . So this test was used .This test found to be more sensitive. Staphylase test reagent and Staphylase control latex was used to perform the test. Latex agglutination (RPLA) kits were provided.we performed the test n kept undisturbed at room temperature for 24 hours.
Temperature of the Italian Lasagne during the time of Ñ€Ð measurement was 23.1°C.
Nutrient information printed on Italian lasagne packet:
Of which saturates :- 14.0g
Of which Total sugars-7.6g
Salt -2.2 g
Saturated fat-14.7 gm
The water activity measured was 0.995
The Ñ€Ð of the food sample was 6.34
Enumeration of potential pathogens:
Nutrient agar plate: -The number of colonies found in 10-3 dilution was 83
i.e:- 8.3Ã-104 cfu/gm
PEMBA plate:-The number of colonies found in 10-3 dilution was 82
i.e;- 8.2 Ã- 104 cfu/gm
VRBG plate:-The number of colonies found in 10-1 dilution was 62
i.e:-6.2 Ã- 103
BPA plate:-The number of colonies found in 10-1 dilution was 1
i.e:- 1.0 Ã-102 cfu/gm.s
Preliminary confirmation tests:
Colonies from PEMBA plates were Gram negative Bacillus with red rod shaped ? . with all the culture. With very few spores.
The given bacillus was catalase positive.
Agar plate was provided pemba n bpa fresh nutrient plate e.
All the colonies from PEMBA, St. aureus culture showed negative result for the oxidase test.
Gram staining and catalase test of presumptive isolates:
The smear prepared from presumptive B. cereus colonies were Gram positive rods with spores
The smear prepared from presumptive St. aureus colonies were Gram positive with cocci.
The presumptive B. Cereus was catalase positive.
The presumptive St.aureus was catalase positive.
In the given API 20 E strip, reagents were added in the well and results were interpreted as per given in the lab manual.
In the given API 50 CHB strip, reagents were added in the well and results were interpreted as per given in the lab manual.