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1 Bacteria Content in Unpasteurized versus Pasteurized Milk and Gram Staining Bacteria

2 Found in Yogurt

3 Bodenhamer, Hannah

4 hannah.bodenhamer@student.csulb.edu

5 California State University, Long Beach (CSULB)

6 Department of Family and Consumer Sciences

7 1250 Bellflower Boulevard, Long Beach Ca 90840

9 ABSTRACT

10 Three experiments were conducted to discovered whether (1) pasteurized milk has more

11 or less bacteria than raw milk (2) an Escherichia coli (E. coli) contamination could be traced to a

12 storage tank containing raw milk or a storage tank containing pasteurized milk and (3) a gram

13 stain could detect gram negative bacteria in a sample of suspect yogurt. Significantly less

14 bacteria grew from a pasteurized milk sample than a raw milk sample. The evaluation of the milk

15 processing plant determined that the tank containing pasteurized milk ready for export was found

16 to contain the same amounts of E. coli as the tank containing raw milk. The raw milk container

17 had approximately 240 colony forming units (CFUs) and the pasteurized milk container had 260

18 CFUs of E. coli. In the gram stain of a suspect yogurt sample, both gram negative (harmful)

19 bacteria and gram positive (probiotic) bacteria were present.

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21 Key Words: Pasteurized, Gram Staining, Contamination, Bacteria

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23 INTRODUCTION

24 The bacterial contamination of dairy products is a worldwide problem. This may be

25 attributed to the highly nutritious composition of milk which therefore serves as a perfect culture

26 medium for many microorganisms (Pal, Mulu, Tekle, Pintoo, & Prajapati, 2016). Some products

27 utilize specific types of bacteria that play key roles in production processes (Morelli, 2014).

28 Other products may undergo treatments such as pasteurization to reduce the number of bacteria

29 present. Pasteurization utilizes a thermal technique involving the rapid heat and cooling of a

30 substance to kill microorganisms and ensure food safety (Monrad, 1895). It is critical that milk is

31 pasteurized before consumption because unpasteurized milk may contain bacteria that can cause

32 serious illness. On the contrary, yogurt is made using lactic-acid producing bacteria that are

33 responsible for the coagulation and fermentation processes that begin the thickening and souring

34 that results in yogurt (Morelli, 2017). One technique that is utilized in food science to

35 differentiate types of bacteria is a gram stain. Gram staining uses a sequence of dyes and

36 chemicals to stain bacteria. Gram positive refers to the bacteria with thick peptidoglycan cell

37 walls that resist decolorization and appear purple under a microscope after the staining process.

38 Gram negative refers to bacteria with a lipid bilayer outer membrane and thin inner layer of

39 peptidoglycan which is much more susceptible to the decolorization effect. The bacteria typically

40 used for the production of yogurt are Lactobacillus, Streptococcus thermophilus, and

41 Bifidobacterium which are all gram positive strains of bacteria. Campylobacter jejuni, E. coli,

42 Listeria monocytogenes, and Salmonella enterica are all common dairy product contaminants

43 and gram negative. These gram negative strains are harmful and can cause serious illness, which

44 emphasizes the importance of reducing contamination of dairy products.

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45 MATERIALS AND METHODS

46 The following lab is completed in 3 parts. In the first part, two samples were obtained

47 from pasteurized and unpasteurized milk. These samples were incubated for 48 hours to allow

48 for bacterial growth. After the incubation period, the two samples were compared and contrasted

49 for bacterial growth. In the second part, multiple samples were taken from different stages of the

50 treatment process at a milk processing plant to determine the origin of an E. coli contamination.

51 These samples were incubated and then evaluated for bacterial growth. In part 3 of the lab, a

52 yogurt sample was tested and evaluated for the growth of harmful bacteria using the gram

53 staining differential process.

54 Part 1: In this section, the number of bacteria found in pasteurized versus unpasteurized milks

55 was compared and contrasted. The materials for this section included 3M Petrifilm Plates for
56 bacterial growth, disposable pipette for sample transferring, and 3M Quick Swabs for sample

57 collection. The Petrifilm Plates were coated in a culturing medium that provided an environment
58 on which bacteria could grow and be identified. First, the station was sanitized with a 70%

59 alcohol solution. Then, a striker was used to light the bunsen burner to a low flame. The first
60 petri dish was then labeled with the date, origin of the sample, and initials of the sampler. Next, a

61 disposable pipette was used to collect 1mL of raw (unpasteurized) milk. This sample was
62 pipetted onto the petri plate, under the top layer of film. Then, the sample was distributed across

63 the plate by pressing a spreader. The same steps were repeated using pasteurized milk. Then,
64 both samples were incubated at 32°C for 48 hours. After the incubation period, both samples

65 contained visible bacterial growth and were easily evaluated based on number and size of
66 colonies.
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67 Part 2: In this section, samples of milk were taken from different points of treatment at a

68 processing plant in order to identify a point of contamination. A 3M quick swab was used to

69 collect wet and dry samples in this section. The first sample came from a wet swab of a 1cm2

70 portion of the dry surface of a refrigerated storage tank in which milk had been stored prior to

71 pasteurization. Next, a dry sample came from a 1cm2 portion of the wet surface of a refrigerated

72 storage tank in which pasteurized milk had been stored prior to packaging. Each sample was

73 labeled with the date, location of the sample, and sampler initials then kept in a cooler containing

74 ice during transportation to an offsite testing center. At the testing center, a work area was

75 sterilized using a 70% alcohol solution and a bunsen burner. Then, two new Petrifilm plates were

76 inoculated using the 3M swabs taken from the milk processing plants. These petrifilm plates

77 contained an agar specifically for the growth of E. coli and each were labeled with the date,

78 location of the sample, and initials. A spreader was pressed onto the film of the petri dish to

79 distribute each sample. Finally, both samples were incubated at 32 degrees celsius for 48 hours

80 then evaluated for bacterial growth. All Petrifilm samples were disposed of using an autoclave

81 for sterilization or incinerator.

82 Part 3: In this section, a yogurt sample suspected of containing harmful bacteria underwent a

83 gram stain to identify the type of bacteria it contained. First, the area was sterilized with a 70%

84 alcohol solution and a bunsen burner was lit to a low flame. Next, a circle was drawn on the

85 underside of a clear slide to indicate where the bacteria sample was located. Then, a bunsen-

86 sterilized inoculation loop was used to collect a yogurt sample by dipping deep into the suspect

87 yogurt container. The inoculation loop was then dipped into 1mL of a phosphate buffered saline

88 (PBS) diluent then shaken within the tube for 5-10 seconds in order to create a diluted yogurt
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89 solution. Next, the inoculation loop was sanitized using the bunsen burner and used to transfer a

90 sample of the yogurt solution onto the slide. The slide, containing two loopfuls of the solution

91 sample, was tilted to spread the sample into a penny-sized shape. Then, the slide passed over the

92 bunsen burner to heat fix the sample. Four chemicals were used to complete the gram stain. The

93 first was a purple stain called Crystal violet. A dropper was used to cover the sample completely

94 then the slide sat for 60 seconds before being rinsed under slow running water. The second

95 chemical was Gram’s Iodine which also sat for 60 seconds before being rinsed with slow running

96 water. Then the decolorization step was conducted using the 95% alcohol solution to flush the

97 slide for 30 seconds, removing all excess dye. After this step, it was important to rinse the slide

98 with slow running water. Finally, the slide was covered in safranin for 60 seconds before a rinse

99 under slow running water. The slide was dried carefully using slightly absorbent bibulous paper.

100 The experiment was then repeated using the normal or good yogurt that did not show any signs

101 of contamination. Both slides were examined under a microscope.

102

103 RESULTS AND DISCUSSION

104 Part 1: Bacteria Sampling of Raw and Pasteurized Milk

105 In the first section, two samples of milk were prepped and incubated, allowing for

106 bacterial growth. Sample 1 contained raw (unpasteurized) milk and had the most visible colonies

107 of bacteria by large. Sample 2 contained pasteurized milk and contained much less visible

108 colonies of bacteria. These observations convey that pasteurized milk contains much less harmful

109 bacteria than raw milk and is thus safe for consumption which can be observed in figure 1. Milk

110 may come into contact with the cow’s udders during the milking process. This introduces
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111 undesirable bacteria from the environment which is harmful to consumers. This is why store-

112 bought milk is always pasteurized for consumer safety.

113 Part 2: Identifying Contamination in Milk

114 In the second section, samples were collected from different points of the treatment

115 process in order to locate the source of E. coli contamination. It was hypothesized that the

116 contamination was coming from the storage tank where raw milk was held prior to processing or

117 the storage tank pasteurized milk was held prior to packaging. For plates with a large amount of

118 growth, the number of colonies is estimated by counting the number of colonies on a square in

119 the center of the agar, then multiplying by 20 (for the 20 cm2 surface area of the Petrifilm). The

120 sample from the tank prior to pasteurization (raw milk sample) had approximately 240 colonies

121 of E. coli growth. The sample from the tank prior to packaging (pasteurized milk sample) had

122 approximately 260 colonies of E. coli growth as seen in table 1. Because the sample of

123 pasteurized milk is similar to the raw milk in bacterial content, it is clear that the pasteurization

124 process is not functioning properly and the bacteria is at an unsafe level for consumption.

125 Part 3: Identifying Contamination in Yogurt

126 As discussed in the introduction, yogurt is fermented with bacteria that is also beneficial

127 to human health. However, E. coli and other harmful bacterium may occasionally end up in

128 yogurt and can result in serious illness to the consumer. Safe yogurt is uniform in color and

129 texture, whereas unsafe yogurt may have air bubbles and separation between the whey and water.

130 It was hypothesized that a sample of yogurt was contaminated with undesirable bacteria. Two

131 samples underwent a gram stain in order to differentiate the harmful bacteria from the normal

132 bacteria used for fermentation. The sample that contained the suspect yogurt and became pink
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133 with the gram stain. This is identified as gram negative (fig. 4) and indicates harmful bacteria.

134 The four chemicals used for the gram stain are Crystal Violet, Gram’s Iodine, 95% alcohol, and

135 Safranin. The Crystal Violet is a purple stain that indicates gram positive bacteria. Gram's Iodine

136 fixes the dye to the cell wall. The 95% alcohol solution initiates decolorization by removing

137 excess dye, leaving only the dye fixed to the cell wall. After these three chemicals, gram positive

138 bacteria will remain purple and can be easily identified under a microscope. Safranin is the final

139 chemical that stains all gram negative bacteria pink. Under a microscope, the normal yogurt

140 appeared purple indicating that it was gram positive and thus did not contain any harmful

141 bacteria. The suspect yogurt appeared pink and purple, indicating there were both gram negative

142 and gram positive bacteria present. The suspect bacteria contained both normal bacteria and

143 harmful bacteria. E. coli, Listeria Monocytogenes, and Salmonella are gram negative bacteria

144 and common contaminants of yogurt (Morelli, 2017). The lactic acid producing bacteria in

145 yogurt are gram positive and are present in both samples.

146

147 CONCLUSION

148 In conclusion, it was discovered that pasteurized milk has considerably less bacteria than

149 unpasteurized milk. Additionally, scientists were able to pinpoint the source of contamination at

150 a milk processing plant to a storage tank where pasteurized milk had been held before packaging

151 and distribution. Finally, two samples of yogurt were tested for contamination. A suspect sample

152 of yogurt was found to contain both gram positive and gram negative bacteria (lactic-acid

153 producing and harmful bacteria). Overall, it was made apparent that it is necessary to conduct

154 frequent testing of contamination in dairy products for the health and safety of consumers.
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155 AUTHOR CONTRIBUTIONS

156 In these virtual experiments, Hannah prepared the lab stations with sanitizer solutions as

157 well as a bunsen burner. In the first and second experiment, Hannah was a lab assistant and aided

158 in the collection of samples as well as the conduction of all laboratory experiments. In the third

159 section, Hannah identified a suspect yogurt sample and collected samples for testing. Hannah

160 performed gram staining procedures and observed the samples under a microscope to

161 differentiate the normal lactic acid producing bacteria from harmful bacteria.

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177 REFERENCES

178
Beveridge, T. (2001). Use of the Gram stain in microbiology. Biotechnic and
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Histochemistry, 76(3), 111–118. https://doi.org/10.1080/714028139
180
Monrad, J. H. (1895). Pasteurization and milk preservation, with a chapter on selling milk.
181
https://doi.org/10.5962/bhl.title.43666
182

183 Morelli, L. (2017). Bacteria in Yogurt and Strain-Dependent Effects on Gut Health. Yogurt in

184 Health and Disease Prevention, 395–410. https://doi.org/10.1016/

185 b978-0-12-805134-4.00023-7

186
Morelli, L. (2014). Yogurt, living cultures, and gut health. The American Journal of Clinical
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Nutrition, 99(5). https://doi.org/10.3945/ajcn.113.073072
188

189 Pal, M., Mulu, S., Tekle, M., Pintoo, S. V., & Prajapati, J. P. (2016). Bacterial Contamination of

190 Dairy Products. BEVERAGE & FOOD WORLD, 43(9). https://doi.org/https://

191 www.researchgate.net/profile/Mahendra_Pal2/publication/

192 308294887_Bacterial_Contamination_of_Dairy_Products/links/

193 57dfcafa08ae0c5b65647e85/Bacterial-Contamination-of-Dairy-Products.pdf

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199 APPENDIX

200 Figure 1. Colony forming units on a Petrifilm Plate from pasteurized milk and raw milk

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202 Figure 2: Comparing samples from pasteurized and raw milk from the processing plant.

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207 Figure 3: Gram positive bacteria under a microscope

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209 Figure 4: Gram negative bacteria under a microscope

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216 Table 1. E. coli Colony Forming Units from Milk Processing Plant

217 Source Approximate Total Colony Forming Units (CFUs)

Raw Milk Storage Tank 240

Pasteurized Milk Storage Tank 260

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