Practical Methodology For Establishing Microbiological Profile Of A Food Sample

Culture Techniques for Microbial Examination of Food

There are various practical methodologies can be used for microbial examination or analysis of foods for example indicator organism’s method, direct examination, culture techniques, enumeration method, laboratory accreditation, and rapid methods to detect specific micro-organism and toxin. The culture techniques provide the opportunity to grow microbial sample in a liquid or on a solid surface. Agar is the polysaccharide with various beneficial properties produced by a species of red algae. Different properties of agar include the ability to form the gel at the concentration or 1 to 2 percent. Such gel is able to stabilize on the high-temperature process in boiling water bath to melt.  It stability to the microbial hydrolysis is another property of agar.

Various types of agar can be used for culture techniques such as plate count agar, MacConkey Broth, lactose broth, glucose agar, blood agar and sucrose agar (Parija, 2014). The main purpose of the media such as nutrient agar, plate count agar, malt extract agar and the dextrose agar for fungi is to provide the nutrition for the growth of microorganisms (Liu, 2018). Culture media techniques provide both qualitative and the quantitative results of microorganisms. This information not only provides data about the presence and absence of microorganism but also provide the information of the number of the organisms. 

For Yoghurt

Sample collection

The food sample containing salmonella should be mixed to make sure the homogeneity before taking the specific amount. The food sample should be pre-enriched at a sample to broth ratio of 1:9 dilutions. The sample should be kept favorable environment. The sample collected for analysis can be the raw or finished product (Andrew and Hammack, 2018).

Dilution

If the count is expected to be higher than 2.5* 10³ per ml, than the decimal dilution should be prepared as:

Shaking the dilution and fresh sterile pipette should be used for each dilution. Pipette one ml of homogenate into a glass tube containing 9 ml of the diluent. One ml is transferred from the first dilution to the second dilution tube, which contains 9 ml of the diluent. This will be repeated to third, fourth, fifth and more glass tubes until the expected dilution is obtained.    

Pour plating

All the Petri plates should be labeled with sample number, date, dilution and other necessary information. One ml of food homogenate pipetted into a petri dish in duplicate. Ten to twelve ml of agar poured into each Petri plate. The media and the dilution should be mixed gently by swirling, clockwise and anticlockwise. The prepared media with dilution is then allowed to set.

Methodology for Examining Yoghurt

Incubation

The prepared dishes are then incubated in an incubator, inverted at 35 degrees or at the desired temperature.

Count colonies

After incubation, bacterial colonies grown on culture median Petri dishes can be counted which are between 30 to 300.

Calculation

In the dishes containing 30 to 300 colonies, the real number in both Petri plates of dilution is then counted with the help of mathematical formula:

                                                                                                                       

Where;

∑ C is the total number of colonies counted on the Petri dishes

N1 is the number of Petri dishes retained in the first dilution

N2 is determined as the number of dishes retained in 2^nd dilution

D is the dilution factor (FSSAI, 2012).

It was developed by Alfred Theodore MacConkey in the 20^th century and was the 1^st solid differential media. MCA or is a differential and selective media which is used to isolate and differentiate the no-fastidious gram-negative bacteria, which belongs to the family of Enterobacteriaceae and genus Pseudomonas.

 Uses of MacConkey Agar

• The MAC agar is used to isolate the gram-negative bacteria.
• This type of agar can also be used to differentiate the lactose fermenting from the lactose non-fermenting gram-negative microorganism.
• It is also used to isolate coliforms and the intestinal disease-causing microorganism in water, biological specimens and dairy product (Engelkirk and Engelkirk, 2008).

Components of MacConkey Agar

• Peptone
• Lactose
• Bile salts
• Sodium chloride
• Neutral red
• Agar
• pH 7.4 ± 0.2 (Atlas, 2010).

Peptone

Peptones are the excellent sources of amino acids, proteins, and peptides in growth media. This mixture of amino acids and protein can be obtained from natural products such as milk, plants and animal tissue.  Peptones provide the important nutrients, nitrogenous factors, and vitamins that are required for the growth of the microorganism.

Lactose

Lactose is the disaccharides or sugar composed of galactose and glucose. The scientific formula for lactose is C₁₂H₂₂0₁₁.  In MacConkey agar, lactose monohydrate is a fermentable source of carbohydrates. It helps to distinguish between the lactose fermenters and non-fermenters. This feature of lactose helps the medium to be selective for lactose fermenters.

Bile Salts

Bile salts are the primary component of bile. In MacConkey Agar, It helps the agar media to be highly selective for gram-negative bacteria and inhibit the growth of gram-positive bacteria.

Sodium chloride

Sodium chloride is an ionic compound also known as salt. It is a metal halide, which composed of sodium and chloride. The molecular formula of sodium chloride is NaCl. This component of MacConkey agar maintains the osmotic regulation in the culture medium.

MacConkey Agar and Its Components

Neutral red

Lactose-fermenting bacteria cause the pH to drop in media, which can be detected by the neutral red component. Neutral red is the indicator of pH that changes in red at the pH below 6.8 and appears as colorless at any pH more than 6.8. The absorbance of neutral red on agar by bacteria results in pink to red colonies.

Agar

Agar is used as the solidifying agent in MacConkey agar. On the solid media, the colonies of bacteria can be seen easily (Batra, 2018).

pH 7.4 ± 0.2

Change in pH is essential to the identification type of bacteria in a culture media.

Coliform appearance on the media is Red.

Selective Media

This type of media is used to isolate a specific group of bacteria. The chemical components used in the culture media prevent the growth of one type of bacteria and promote the growth of another. The examples of selective media are MSA (Mannitol Salt Agar), MacConkey and EMB (Eosin Methylene Blue Agar).

Differential media

It distinguishes among the groups of microorganisms that biologically and morphologically related. The chemical compounds of this media produce a change in the appearance of microbial growth. Examples:  EMB, MSA, and Macconkey (Semmelweis, 2014).

Enriched Media

This culture media is supplemented with the high amount of nutritious material like serum, yeast extract, and blood for microbial cultivation.

A basic principle of this media is to control the nutrients and the culture conditions (temperature, light, pH and air supply) for the specific type of species (Vanmeter, Hubert, and Vanmeter, 2013).

The TRS wholesale Co ltd recalls the product TRS turmeric powder because they found that the product might contain bacterial species salmonella.

Salmonella is a rod-shaped bacteria of family Enterobacteriaceae commonly found in the intestinal tract of humans. The salmonella infection is the foodborne disease that caused by the consumption of eggs, raw meats, and contaminated dairy products. Particularly in the United States, nearly 19000 people are affected by salmonella food poisoning every year.  The salmonella associated food poisoning includes the symptoms like diarrhea, abdominal pain, nausea, and vomiting.

The company has mentioned that only TRS turmeric powder is assumed affected and no other product.

Salmonella infection is the 2^nd most common infection in the United States. In 2009 more than 7000 cases have been confirmed.it is the major health global health concern accounting for 155,000 deaths per year (Eng et al. 2015). The salmonella associated infection occurs when an individual eats foods that contaminated with faces of animals. Most of the infection caused by salmonella occurs due to eating foods such as eggs, poultry, and meat. Salmonella can also infect other foods such as raw milk, raw meats, and contaminated water.

Selective and Differential Media

It can various health issues in humans such as cramps, fever, abdominal pain, and diarrhea. The typical symptom caused due to salmonella infection appears between 6 to 72 hours of consuming contaminated eatables. It lasts for three to seven days without treatment. The additional symptoms of this type of infections are bloody diarrhea, vomiting, headache and boy aches (Mayoclinic, 2018).

Fever

The typhoidal salmonella may also cause typhoid fever, this type of infection is rare and caused by a species of salmonella called Salmonella typhi, humans are the only carrier of this type of infection. The typhoid is a fever of 105 F, includes symptoms like weakness, pain, coughing, lethargy, nosebleeds, enlarged organs, and delirium. Typhoid fever is the life-threatening disease may lead to death if remain untreated (Crump, 2010).

Other complications associated with salmonella infection

Salmonella poisoning related complications mostly affect young children and the people 65 years old or more. Reactive arthritis is the possible complication of this type of infection

Reactive arthritis can occur in 2 to 15 percent of salmonella patient. The symptoms of this disorder include Inflammation of eyes, joints, or urinary or reproductive organs. The symptoms of reactive arthritis occur eighteen days after infection.  

Focal infection: this type of infection occurs when the bacteria reaches top body tissue and cause illness like arthritis or endocarditis. It is also caused by typhoidal salmonella (Clerk, 2018).

1. Smear preparation: A very thin smear of the sample is prepared on a glass slide, heat fix and allows it to cool.
2. The air-dried and heat fixed smear is then flooded with crystal violet reagent for one minute.
3. The glass slide is then washed in tap water for two seconds.
4. After that, the slide is flooded with gram iodine and wait for one minute.
5. The iodine-flooded slide is then washed under running tap water for two minutes.
6. The decolorization agent Acetone- alcohol is then applied on the slide.
7. After applying decolorizing agent on the slide for 10 to 15 seconds, safranin is flooded on that glass slide and left to dry for thirty seconds to one minute.
8. The dried slide is then washed under running tap water until there is no color appears in an effluent. After that blot, dry the slide with absorbent paper.
9. Oil immersion is then applied on the slide and observe under the microscope for results (Bruckner, 2016).

Results

• The gram-negative bacteria in a sample appeared as pink/red.
• The gram-positive bacteria have appeared as blue/ purple.

1. Structure of bacterial cell wall

The layers of a cell envelope found between the cytoplasmic membrane and capsule are known as the cell wall. The main function of these call walls is to provide rigidity and integrity to the cell. These layers are composed of peptidoglycan and teichoic acid.

1. Peptidoglycan: it is 20 to 80 nanometre in thick and forms nearly 90 percent of the total dry weight of bacteria. These are the polymers of NAM (N-acetylmuramic acid) and NAG (N- acetylglucosamine). Long strands of peptidoglycan attached with NAM. These polymers are more highly cross-linked in case of gram-positive cells than the gram-negative bacterial cell. This peptidoglycan is the target of antimicrobial drugs such as penicillin, which cleaves the NAM_NAG bond.
2. Polysaccharides: the lipoteichoic acids or LTA are found in gram-positive bacteria only. These polysaccharides are present in entire peptidoglycan later. These call wall structure can serve as the antigenic determinants.
3. Lipopolysaccharides (LPS): these structures are found in gram-negative bacteria only. Lipopolysaccharides are composed of lipid A that attaches LPS to the outer layer. These polysaccharides appear on the cell surface and serve as the antigenic determinant.
4. Porins: porins are the Transmembrane proteins made of a trimer with three subunits. The main function of these proteins is to transfer molecule across the membrane (Saltmann and Holst, 2013).

                                   

                         Figure 1 Structure of Bacterial cell wall of gram-positive and gram-negative bacteria (Bruslind, 2018)

1. Staining with the water-soluble dye named crystal violet. After that, gram iodine is applied to form a crystal violet-iodine complex.
2. Ethyl alcohol as decolorization agent added to the given sample. This dehydrates, shrink and tightening the peptidoglycan layer. The complex is unable to penetrate the peptidoglycan layer and trapped in the gram-positive bacterial cell. The outer membrane gram-negative bacterial cell degraded. The peptidoglycan layer is now not able to retain the complex and the color is then lost.
3. Safranin is than added to the sample. Due to the lighter color of safranin not able to disrupt the purple color in gram-positive cell and the cells of gram-negative bacteria stained red.

                                             

                                                                  Figure 2 Steps of gram staining (Laboratoryinfo, 2016)

A notifiable disease is any disease or infection of animals and humans, that needs to report to the government authority (Health direct, 2017). Various diseases are considered notifiable by the Public Health England (PHE) such as acute encephalitis, anthrax, botulism, cholera, diphtheria, enteric fever, food poisoning, infectious bloody diarrhea, smallpox, tetanus, and tuberculosis. In the given statement, the government organization wanted to identify any disease that causing health issue for people, needs to be reported as soon as possible. They asked to report any suspicious outbreak of a particular disease after 1968. The accuracy of the diagnosis of the disease or infection is not their priority. For the foodborne diseases data of food that caused health issues and the responsible microorganism caused that, the disease needs to be identified and reported. The food poisoning related issues are caused due to the defect in the manufacturing of that product.  By identifying the particular type of disease or infection caused by eating specific type food, helps to stop the manufacturing of that product. 

Discussion of Statements Related to Food Microbiology

Report & monitor notifiable disease.

The notifiable diseases are monitored by the state government and national authority. There are some duties and responsibility of the laboratories and medical practitioners set by the government to regulate and report any outbreak of the disease and the causative agent associated with illness.

According to public health (control of disease) Act 1984 and Health protection (notification) regulations 2010 the statutory duties for reporting the notifiable diseases: 

• The PMPs (registered medical practitioners) have the responsibility to report to an appropriate authorized person to their local council or to the health protection team.
• The RMP is that needs to complete the notification form just after the diagnosis of a suspected notifiable disease. They do not need to wait for the laboratory results or confirmation of a suspected infection or the contamination before reporting.
• The completed form is then sent to the authorized officer within three days, or report them directly within one day (Public health England, 2010).
• If the case needs urgent action, this can be reported on the phone call, or by e-mail and letter. If the medical practitioners need help, then they can contact the local health protection team. They can find the contact information by postcode lookup.
• According to the health protection legislation (England) guidance 2010 if the RMP believe that the case has been already reported by another RMP, it is not essential to report it again (Department of health, 2010)

Notifiable organisms        

All the authorized officer need to pass the RMP’s entire notification to Public health England within three days of a case is reported and within one day in emergency cases. The notifiable organism that considered notifiable is Bacillus anthracis, Bordetella pertussis, Borrelia species, Brucella species, Burkholderia mallei, Clostridium botulinum, Ebola virus, Mumps virus and many more (Public health England, 2010).

Subscribe to the report

The last step is subscribed to report. After receiving the notification from RPM the Public Health England, publish the report on the received laboratory notification. PHE’s data management section publishes the report analysis of trends per week.  

Foodborne disease is the most common problem n all over the world caused by consuming the food that is contaminated with the harmful microbial pathogens or agents such as bacteria and virus. Foodborne illnesses are considered a threat to the public health, which responsible to cause 2.2 million death work wide, and 500 hundred deaths in the United Kingdom. Particularly in the UK estimated 1 million people affected by the foodborne disease each year. The government spends nearly 1.5 billion euros per year.  Study conducted by Lund (2015) shows that particularly in the UK in 2009 nearly 280, 400 foodborne illness case has been reported due to the infection caused by clostridium perfringens pathogen, Norovirus accounts for 79,570 cases, salmonella non-typhoidal associated infection were 33,130, cases of E. coli infection were recorded 9886 and Campylobacter associated cases were reported 280, 400 (Lund, 2015).

Campylobacter is the genus of gram-negative bacteria, which appears comma-shaped and motile. Commonly found in raw meat, raw chicken, and turkey. It can also be found in milk, water, occasionally in mushroom and shellfish. Two different species (C. jejuni and C. coli) of Campylobacter cause most of the infections in the UK. The disease caused by Campylobacter is campylobacteriosis that can occur within two to five days. The symptoms of Campylobacter infection are nausea, vomiting, diarrhea, and bloody diarrhea. Theodore Escherich reported the first concerning campylobacter in 1986, after that it was identified in 1906 in a pregnant sheep. After that various case has been recorded worldwide. Campylobacter species are accounts for major bacterial infection in the UK.  In 2009, more than 64,000 cases were reported in Scotland and England and increasing by 30% and 14 % per year (Strachan and Forbes, 2010). These microorganisms can survive and grow in between 37 to 42-degree centigrade. 

Food  Standards Agency (FSA) conducted a cross-sectional study among the people of United Kingdom and found that the knowledge about campylobacter was lower than expected, the survey revealed that ninety percent people heard about the microorganisms like Salmonella and E. coli but only 28 percent were aware of campylobacter. The increase the knowledge and to reduce the campylobacter associated illness FSA launched a health campaign named Acting on Campylobacter Together or ACT (Persons, 2014).

For this campaign, FSA brought together the representatives from various sectors such as government, caterers, retailers, processors and poultry producers, and consumers. The five-stage plan has been prepared to tackle the problem from poultry farm to fork:

1. Tackling the issue at source (farms)
2. Reducing the risk at the processing stage
3. Maintain the low temperature while transporting the fresh poultry.
4. In-store ‘cook in bag’ whole meat, decrease the need for consumers to handle the chicken.
5. Do not wash sticker on chicken.

Effects of this five-point plan show good results, it reduces the campylobacter mediated infection was reduced from 11 % to 7 % (Whitworth, 2015).  In the starting months of 2017, the results of the ACT shows a great reduction in the level of campylobacter infected chicken from 20.3 percent to 7%. The Co-op results for Q1 2018 revealed that zero percentage of chicken had contaminated. According to an article published in Poultry, the world revealed that from the seventh month to 9^th month of 2015, fifteen percent of chicken testes positive for the maximum level of contamination, which is down from 22 percent (Poultry world, 2015).

References

Andrew, W. H., and Hammack, T. S. (2018). BAM: food sampling/preparation of sample homogenate. Available from: https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm063335.htm Accessed 28 July 2018.

Atlas, R. M. (2010). Handbook of microbiological media. 4^th ed. London, New York: CRC Press. p. 933.

Batra, S. (2018). Preparation of Macconkey broth medium in the laboratory. Available from: https://paramedicsworld.com/microbiology-practicals/preparation-macconkey-broth-medium-laboratory/medical-paramedical-studynotes#.W12KvNUzbIU Accesses 29 July 2018.

Bruckner, M. Z. (2016). Gram staining. Available from: https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html Accessed 28 July 2018.

Bruslind, L. (2018). Bacteria: cell walls. Available from: https://library.open.oregonstate.edu/microbiology/chapter/bacteria-cell-walls/ Accessed 29 July 2018.

Clerk, M. (2018). Salmonella. Available from: https://www.foodborneillness.com/salmonella_food_poisoning/  Accessed 28 July 2018.

Crump, J.A. and Mintz, E.D., 2010. Global trends in typhoid and paratyphoid fever. Clinical Infectious Diseases, 50(2), pp.241-246.

Department of health (2010). Health Protection legislation (England) guidance 2010. Available from: https://webarchive.nationalarchives.gov.uk/20130105053557/https://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/@ps/documents/digitalasset/dh_114589.pdf Accessed 29 July 2018.

Eng, S.K., Pusparajah, P., Ab Mutalib, N.S., Ser, H.L., Chan, K.G. and Lee, L.H. (2015). Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science, 8(3), pp.284-293.

Engelkirk, P. G., and Engelkirk, J. L. D (2008). Laboratory Diagnosis of infectious diseases: essentials of diagnostic microbiology.  Philadelphia, PA: Lippincott Williams & Wilkins, pp. 134-135.

FSSAI (2012). Manual of methods of analysis of foods. Available from: https://old.fssai.gov.in/Portals/0/Pdf/15Manuals/MICROBIOLOGY%20MANUAL.pdf Accessed 28 July 2018.

Health direct (2017). Notification of illness and disease. Available from: https://www.healthdirect.gov.au/notification-of-illness-and-disease Accessed 29 July 2018.

Laboratoryinfo (2016). Gram Staining: principle, procedure, interpretation, and animation. Available from: https://laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-animation/ Accessed 29 July 2018.

Liu, S. (2018). All about agar. Available from https://www.sciencebuddies.org/science-fair-projects/references/grow-microbes-agar Accessed on 28 July 2018.

Lund, B.M., 2015. Microbiological food safety for vulnerable people. International journal of environmental research and public health, 12(8), pp.10117-10132.

Mayoclinic (2018). Salmonella infection. Available from: https://www.mayoclinic.org/diseases-conditions/salmonella/symptoms-causes/syc-20355329 Accessed 28 July 2018.

Parija, S. C. (2014). Textbook of microbiology & immunology. 2^nd ed. India: Elsevier Health Science, pp. 34-35.

Persons, H. (2014). UK food standards agency kicks off anti campylobacter campaign. Available from: https://www.foodsafetymagazine.com/news/uk-food-standards-agency-kicks-off-anti-campylobacter-campaign/

Poultry world (2015). Level of campylobacter in British chicken down. Available from: https://www.poultryworld.net/Meat/Articles/2015/11/Level-of-campylobacter-in-British-chicken-down-2722743W/ Accessed 29 July 2018.

Public health in England (2010). Notifiable diseases and causative organisms: how to report. Available from: https://www.gov.uk/guidance/notifiable-diseases-and-causative-organisms-how-to-report Accessed 29 July 2018.

Saltmann, G. and Holst, O. (2013). The bacterial cell wall. 3^rd ed. Heidelberg, New York: Springer Science & Business Media.

Semmelweis (2014). Enrichment, selective and differential culture media. Available from https://semmelweis.hu/mikrobiologia/files/2014/05/Omid_media_3.pdf Accessed on 28 July 2018.

Strachan, N.J. and Forbes, K.J., 2010. The growing UK epidemic of human campylobacteriosis. The Lancet, 376(9742), pp.665-667.

Vanmeter, K. C., Hubert, R. J., and Vanmeter, W. G. (2013). Microbiology for the healthcare professional.  New York, US: Elsevier Health Sciences. Pp. 85-86.

Whitworth, J. J. (2015). FSA to Publish New Batch of Campylobacter results. Available from: https://www.foodnavigator.com/Article/2015/11/17/Retailer-progress-on-tackling-Campylobacter-on-chicken Accessed 29 July 2018.