Experiment on the Presence of Microorganisms in a Milk Sample

Assignment title:

A quantitive and qualitive investigation into the presence of microorganisms in a milk sample by the use of a serial dilution

1.0. Introduction

Microbiology relates to the study of microbes or microorganisms, which are too small to be visible by the naked eye, including simple life-forms such as; fungi, algae, protozoa, bacteria, viruses, and archaea. These play important roles in the contribution of climate change, food spoilage, disease control, biotechnology, nutrient cycling, and biodegradation.
When bacteria are viewed under a microscope, they have three distinct shapes, ‘Bacilli’ – rod-shaped, ‘Cocci’ – sphere shaped, and ‘Spirilli’ – spiral shaped. All bacteria cells are found to have; a cell wall, nucleoid, capsule, cytoplasm, ribosomes, plasmid, plasma membrane (as shown in image 1).

Image 1: Bacteria Cell Structure (Davidson, 2015)

Fungi are either multicellular or unicellular (dependant on the environmental conditions), and structurally made up of fine threads called ‘hyphae’, which when intertwine, make tangles of filaments known as ‘mycelium’. The cells also contain; the endoplasmic reticulum, the Golgi apparatus, and mitochondria. However, unlike plant cells, fungi do not have chlorophyll or chloroplasts (as shown in image 2).

Image 2: Fungi Cell Structure. (Structure and Physiology of Fungi, 2016)

Viruses are constructed of a capsid or head region (made of glycoproteins and proteins), in which is contained its genetic material. This allows the viruses to puncture the host cells through membrane fusion. Additionally, some viruses contain a tail region, which is an elaborate protein structure, allowing them to bind to the surface of the host cell (as shown in image 3).

Image 3: Virus Cell Structure. (General Characteristic of Viruses, n.d.)

Protozoa are unicellular eukaryotes, in which the nucleus is enclosed in a membrane, as well as containing other important organelles inside the cytoplasm, such as the flagella, cilia, and pseudopodia.
Due to these microorganisms existing as a diverse group, they can vary in shape and size (as shown in image 4).

Image 4: Cellular features and structure of various types of Protozoal organisms. (Ashwathi, n.d.)

This lab report comprises of an investigation into the different microorganisms present in a diary product – milk. Some of these may include; lactic acid bacteria, coliforms, clostridium, streptococcus, bacillus cereus, salmonella, Escherichia coli O157, and campylobacter. This experiment is carried out by a serial dilution. A serial dilution is used to measure the quantity of bacteria present in a food sample, by performing a series of sequential dilutions to reduce a dense culture of cells to a more usable concentration (Serial Dilution in Microbiology: Calculation, Method and Technique , n.d.).

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Each dilution reduces the concentration of the bacteria present by a specific amount, thus allowing the calculation to determine the quantity of bacteria present at the start (Keler, Balutis, Bergen, Laudenslager, & Rubino, 2010). 
Carrying out tests to identify which microbes are present in a food sample is highly important to; determine the sanitary quality of the products, prevent spoilage, and destroy any pathogenic microorganisms that may be present (Blodgett, 2009). However, in various dairy products such as cheese and yogurt, some microorganisms are desirable to produce essential chemical changes, in order to create the desired aromas and flavours.
One method used to help to identify the type of bacteria present is gram staining. This involves a bacterial smear being placed upon a plate, left to dry and then stained with crystal violet (made of positively charged coloured ions). It is then applied with Gram’s iodine, creating a larger molecule. A decolouriser is further added, which for Gram positive bacteria, is unable to penetrate the peptidoglycan layer, remaining trapped in the cell. In contrast, Gram negative bacteria is unable to retain the crystal violet-iodine and the colour dissipates.
Agar is highly important in the practice of microbiology as, unlike regular gelatine, it cannot be eaten by bacteria. Agar can be used to feed and grow microorganisms, as it provides the nutrients needed. Furthermore, there are various kinds of Agar used (blood agar, chocolate agar, MacConkey agar, nutrient agar) depending on the results needed. This can be dependent on the type of microorganisms, pH levels, and Gram-negative or Gram-positive.
Some of the microorganisms that grow on Plate Count Agar (PCA) and Baird Parker Agar include; Bacillus, Escherichia Coli O157, Lactobacillus, and Staphylococcus. Additionally, Tryptone Bile Ager and Violet Red Bile Glucose Agar, permit the growth of microorganisms such as; Escherichia Coli O157, Klebsiella pneumoniae, Salmonella Choleraesuis, and Shigella Flexneri.
 

Counting the colonies of microorganisms that grow on agar plates is essential to determine the concentration of microbes in a sample. This is achieved manually or automatically by the use of various methods; The Membrane Filter, The Miles and Misra Methods, The spread Plats, and The Pour Plate. Additionally, OpenCFU and NICE are both software’s used for counting, in order to counteract human error.
Using aseptic technique is highly important when working with microorganisms, to prevent any contamination or infection, whilst also helping to isolate and propagate bacteria.

Method
Equipment:

 - 6  test tubes
– 2  1ml pipette
– 1  Pipette filler
– 1  Bottle of MRD (Maximum Recovery Diluent)
– 6 x Plates
– Violet Red Bile Glucose Agar (VRBGA)
– Tryptone Bile Agar (TBX)
– Baird Parker Agar (BP)
– Plate Count Agar (PCA)

• The original milk sample was placed into the first test tube using a pipette filler.
• The second test tube was then filled with 9ml of MRD, using a pipette filler, and 1ml of the original milk sample, using a 1ml pipette, and swirled to ensure that it was fully diluted.
• This process was repeated a further 4 times, each taking a sample from the previous test tube (refer to Image 5).
• 0.1ml of the milk dilution from each sample was measured using a 1ml pipette and placed onto an Agar plate.
• The Agar was then poured onto the plate with the milk and swirled gently, to ensure fully mixed.
• This process was then repeated for the remaining milk samples and Agar.
• Once completed, the plates were left to set, and turned upside down to allow airflow and bacteria growth.
• The plates were then left between 3-5 days at 40c, to allow any bacteria growth, if present.
• After the set time period, the plate was then counted to determine the number of colonies. Only the plate with 30-100 colonies on were counted – this was 1 in 1,000 dilution.
• The colonies were counted by using a colony counter machine, counting automatically each time the colonies were marked off.
• The data was then recorded and used to calculate the quantity of microbes in the milk sample
•  To determine which colonies were present, gram staining was used. This occurs by removing a sample and using the results to determine which colonies are present (refer to Image 6).

Image 5: Serial dilution of milk (Kenneth, 2017).
 

Image 6: Gram Staining (Tankeshwar, 2015)

Results
 

Image 7: PCA showing the quantity of colonies present in the original sample of milk. 

      Image 8: PCA showing the quantity of colonies                                                                                     present in 1 in 10 dilution in a sample of milk.

      Image 9: PCA showing the quantity of colonies                                                                                     present in 1 in 100 dilution in a sample of milk.

      Image 10: PCA showing the quantity of colonies                                                                                     present in 1 in 1000 dilution in a sample of milk.

      Image 11: PCA showing the quantity of colonies                                                                                     present in 1 in 10 000               dilution in a sample of                                                                                     milk.

      Image 12: PCA showing the quantity of colonies                                                                                     present in 1 in 100 000 dilution in a sample of                                                                                     milk.

      Image 13: TBX showing the presence of E.coli                                                                                     in a milk sample.

      Image 14: VRBGA showing the growth of                                                                                      enterobacteria in a milk sample.

      Image 15: BP the growth of Staphylococcus                                                                                      Aureus in a milk sample.

Discussion
The Plate Count Agar (PCA) results show 6 various dilutions of milk samples using MRD. There was found to be 210 colonies in the milk dilution sample of 1 in 1,000. This is then multiplied by the dilution factor itself; 210  1000 = 210,000cfu/ml. The results shown are from a chosen plate, with 30-300 colonies present, which had a dilution of 1 in 1,000. This shows that the milk tested is not fit for sale, as the UK legal limit for microbes in milk is 100,000cfu/ml.
The different Agars used in this experiment to test for bacteria, include; E.coli (originating from faeces), Staphylococcus Aureus (originating from skin) and the awareness of yeast and moulds.
Violet Red Bile Glucose Agar (VRBGA) aids in the detection of Enterobacteriaceae in food and dairy products. The results given portrayed E.coli growth in the sample milk. This was visible by the presence of pink to red colonies with bile precipitate.
Tryptone Bile Agar (TBX), also showed that there was E.coli growth present in the milk sample. This could be seen by blue colonies present on the plate.
(Viçosa, Moraes, Yamazi, & Nero, 2010) Baird Parker Agar (BP) test exhibited the growth of Staphylococcus Aureus, by the presence of grey to black shiny convex colonies, with a ‘halo’ zone of clearing. This agar also detects the presence of any moulds or yeasts, however, there was not found to be any present in the sample tested. 
Errors may have occurred in this experiment due to; not reading the measurements accurately on the pipettes, unsterile equipment, using equipment incorrectly, the presence of bacteria and microbes present in the air when transferring the milk from the test tubes, and also the agar to the plates.
This could have been improved by ensuring the use of sterile equipment, wearing lab coats and hairnets, and working in a sterile lab in order to prevent any cross contamination. Additionally, conducting further serial dilutions of milk and placing the samples on PCA, would enable the comparison between the different sets of plates. Furthermore, repeating the process 3 times, of placing the milk samples in fresh PCA and counting colonies, will aid to achieve an average, whilst also ensuring more accurate results. 

Conclusion
This experiment showed that the cfu/ml value of the original sample of milk was 210,000cfu/ml. The sample tested would be deemed unfit for sale due to the UK legal limit for microbes in milk being 100 000cfu/ml.
There was also found to be various different microorganisms present in the milk sample, including E. coli and staphylococcus aureus. This could lead to severe consequences for the industry, resulting in the loss of profit and money, whilst also facing the possibilities of prosecution if the product caused a consumer illness. The bacteria present in the milk sample (E.coli and Staphylococcus Aureus) tested have the ability to cause a person grave illness, often resulting in the need for hospital treatment. In contrast to this, there was not found to be any yeasts or mould present in the sample. These would be shown on the PCA and BP agars, by the presence of white colonies.

References

• Ashwathi, P. (n.d.). Examples of Protozoa. Retrieved from Biology Discussion: http://www.biologydiscussion.com/protozoa-2/examples/examples-of-protozoa-with-diagram/49967
• Davidson, M. W. (2015, November 13). Cell Biology and Microscopy Structure and Function of Cells and Viruses. Retrieved from Molecular Expressions: https://micro.magnet.fsu.edu/cells/bacteriacell.html
• General Characteristic of Viruses. (n.d.). Retrieved from Spark Notes: https://www.sparknotes.com/biology/microorganisms/viruses/section1/
• Serial Dilution in Microbiology: Calculation, Method and Technique . (n.d.). Retrieved from Study.com: https://study.com/academy/lesson/serial-dilution-in-microbiology-calculation-method-technique.html
• Keler, C., Balutis, T., Bergen, K., Laudenslager, B., & Rubino, D. (2010). Serial dilution simulation lab. The American Biology Teacher, p305.
• Blodgett, R. J. (2009). Planning a serial dilution test with multiple dilutions. Food Microbiology, p421-p424.
• Structure and Physiology of Fungi. (2016). Retrieved from Cliffs Notes: https://www.cliffsnotes.com/study-guides/biology/microbiology/the-fungi/structure-and-physiology-of-fungi
•  Kenneth. (2017, October). Serial Dilution of milk samples. Retrieved from ResearchGate: https://www.researchgate.net/figure/Serial-dilutions-of-milk-samples-in-10-test-tubes-containing-9-ml-of-sterilized-distill_fig1_320780727
• Tankeshwar. (2015, February 2). Gram Staining: Principle, Procedure and Results. Retrieved from Microbe Online: https://microbeonline.com/gram-staining-principle-procedure-results/
• Viçosa, G. N., Moraes, P. M., Yamazi, A. K., & Nero, A. L. (2010). Enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese: An evaluation of Baird-Parker agar, Rabbit Plasma Fibrinogen agar and the Petrifilm™ Staph Express count system. Food Microbiology, p447-p452.