UNIVERSITI MALAYSIA TERENGGANU

PUSAT PENGAJIAN SAINS DAN TEKNOLOGI MAKANAN

LAB REPORT 1

STM 3103 (FOOD SAFETY MANAGEMENT)

EFFECT OF ENVIRONMENTAL FACTORS ON THE GROWTH OF MICROORGANISMS

Prepared by:

Group 17 [Bachelor of Food Science (Foodservice & Nutrition)]

CHONG HAN HUI (UK28095)

CHONG POOI YAN (UK29588)

NUR ARIFAH SABRINA BINTI MD ZAKI (UK28094)

NURUL NAJIHAH BINTI CHE MOKHTAR (UK28083)

Objective

To determine the effect of temperature, osmotic pressure, pH and oxygen presence

on the growth of Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus,

Escherichia coli and Salmonella.

Introduction

There are two factors required for bacterial growth, environmental factors

affecting growth and also source of metabolic energy to bacteria survive, growth and

reproduce. Environmental factors affecting growth of bacteria are nutrients, pH of

the medium, gaseous requirement, temperature, light, ionic strength and osmotic

pressure. The source of metabolic that affecting the growth of microorganism are

reproduction and growth curve. Different types of bacteria have different optimum

temperature, optimum temperature is temperature where maximal growth of

microorganism. The optimum growth temperature determines its classification as a

thermophile, mesophile or psychrophile (Forsythe, 2008). The solute concentration

of a solution effects the osmotic pressure that exerted across the cytoplasmic

membrane of a microorganism. According to Archunan (2004), the cell wall

structures of bacteria and other microorganism make them relatively resistant to

change in osmotic pressure, but extreme osmotic pressure can result in the death.

Different microorganisms have different optimum pH most spoilage bacteria grow

best near neutral pH, pathogenic bacteria even more narrow in tolerance range or

near neutral, while yeast and mold have much greater intolerance to acidic. The

optimum pH range is usually quit narrow so that small changes in pH can have large

effects on the growth rate of the organism. Another factor that greatly influences

microbial growth rates is concentration of molecular oxygen. Microorganism can be

classified as aerobes, anaerobes, facultative anaerobes, or microaerophiles based on

their oxygen requirements and tolerances. Areobic microorganisms grow only when

oxygen is available, while anaerobic microorganisms grow in absence of oxygen.

Facultative anaerobes are capable of both fermentation and respiratory metabolisms

but microaerophiles required oxygen but exhibit maximal growth rates at reduced

concentrations because higher oxygen are toxic to these organism. (Archunan, 2004)

Result

Table 1: Effect of temperature on microbial growth

Temperature

/Microorganism

5 0C 25 0C 37 0C 50 0C

P. aeruginosa - +++ ++++ -

B. subtilis - ++++ ++++ +++

S. aureus - +++ ++++ -

E.coli - +++ ++++ ++

Salmonella - +++ ++++ -

Table 2: Effect of osmotic pressure (salt) at 37 0C

Percentage of salt

/Microorganism

0% 1% 5% 10%

P. aeruginosa ++++ +++ +++ ++

B. subtilis ++++ +++ ++++ -

S. aureus ++++ +++ +++ ++

E. coli - ++++ +++ -

Salmonella +++ ++++ ++ -

Table 3: Effect of osmotic pressure (sugar) at 37 0C

Percentage of

sugar/Microorganism

0% 1% 10% 20%

P. aeruginosa ++++ +++ +++ ++

B. subtilis ++++ ++++ +++ -

S. aureus ++++ +++ ++ ++

E. coli - ++++ +++ -

Salmonella ++++ ++++ - -

Table 4: Effect of pH at 37 0C

pH/

Microorganism

3.0 5.0 7.0 10.0

P. aeruginosa - +++ ++++ ++

B. subtilis - ++++ ++++ ++

S. aureus - ++ ++++ +++

E. coli - +++ ++++ -

Salmonella - +++ ++++ +++

Table 5: Effect of oxygen at 37 0C

pH/

Microorganism

With paraffin oil layer Without paraffin oil layer

P. aeruginosa - ++++

B. subtilis +++ ++++

S. aureus +++ ++++

E. coli +++ ++++

Salmonella +++ ++++

Discussion

The microorganisms used in this experiment are Pseudomonas aeruginosa,

Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Salmonella. Based on the

result, all the microorganisms such as P. aeruginosa, B. subtilis, S. aureus, E. coli and

Salmonella showed the best growth at temperature of 37˚C. At temperature of 5˚C,

all the microorganisms did not showed any sign of growth on the growth culture.

This is because germs and bacteria grow and multiply very fast in the temperature

range of 5˚C to 57.2˚C which also known as temperature danger zone. Thus, under

temperature of 5˚C, the growth of microorganisms will be slow down and prohibited.

37˚C is right in the middle of the temperature danger zone where the

microorganisms grow the fastest (Paster, 2007). Higher temperature inactivates

certain essential enzyme systems associated with cell division (Davies & Board,

1998). P. aeruginosa showed no growth based on the result obtained, this is because

the optimum temperature for P. aeruginosa is at 37˚C and it is able to grow as high

as 42˚C which also known as the maximum temperature for growth (Todar, 2009).

Besides, Salmonella also showed no growth at temperature of 50˚C because the

maximum temperature for Salmonella is at 45˚C and minimum temperature is at 9˚C

which mean the growth is slow down or prohibited out of this range of temperature

(Vanderheijden, 1999). E. coli act as a mesophilic bacteria, did not multiply at storage

temperatures below 5˚C (Davies & Board, 1998). This theory explained the reason

why we observed there was no sign of growth of E.coli at temperature of 5˚C. For the

microorganism of B. subtilis, it showed no growth at temperature of 5˚C and 50˚C, it

is because the range of growth of temperature of B. subtilis is from 20˚C to 50˚C

(Srivastava, 2003). According to Srivastava (2003), the maximum temperature of S.

aureus is 45˚C but it showed moderate growth at temperature of 50˚C which already

beyond the maximum temperature. The result obtained is opposed the theory given.

This situation can be explained by the mishandling of procedure during the

experiment such as human negligence or the incorrect set-up of incubator’s

temperature.

Based on the result observed, Salmonella showed depleted growth sign along

with the increment of percentage of salt and sugar used in the growth culture. When

Salmonella was grown in a glucose-salts medium, additions of NaCl, sucrose, and a

mineral salts mixture produced comparable growth inhibition at all water activity

levels tested (Lund et al, 1999). S. aureus grow best at 0% of salt percentage but still

continues showed the sign of growth at 10% of salt percentage. This is because S.

aureus is salt tolerant and grows at concentrations greater than 10 percent of NaCl

(Archunan, 2004). In the exceptional of S. aureus which can survive at 10% of salt

percentage, the other microorganism such as P. aeruginosa, B. subtilis, E.coli and

Salmonella showed no growth in the medium which contained of 10% of salt

percentage. This is because when NaCl used in higher concentration, it will exert a

high pressure which has the drying effect on the cells due to plasmolysis, ultimately

causing the death. However, halodurics, halophiles tolerate the high osmotic

pressure (Soni, 2007). Salt is highly preservative when its concentration is increased,

and levels of 18-25% in solution generally will prevent all growth of microorganisms

in food (Potter & Hotchkiss, 1998).

Adding high concentrations of sugar, such as sucrose, to a solution lower the

availability of water (Archunan, 2004). B. subtilis and Salmonella showed no growth

based on the result observed whereas the S. aureus showed depleting growth curve

along with the increment of sugar concentration from 0% to 20%. Bacteria cannot

row in high concentration of sugar and salt, yeasts can grow in fairly high

concentrations whereas molds grow in the highest concentrations of sugar (Roday,

1999). Salt and sugar is the most common substances used to create hypertonic

environment for microorganism and prevent them from growing. When a cell is

place in a hypertonic solution, water flows out of the cell into the surrounding

solution causing the cell to shrink and lose its turgidity. P. auruginosa, S. aureus and

E. coli showed the sign of growth at 20% of sugar because usually 70% sucrose in

solution will only stop the growth of all microorganisms in foods (Potter & Hotchkiss,

1998).

In the third experiment we use pH 3.0, 5.0, 7.0 and 10.0 to determine the

effect of pH on microorganism growth. From the result, all the microorganisms

showed no growth in pH 3.0 and the broth are transparent, have no sediment and

foam. This showed that all the tested microorganisms are not acidophilic.

Acidophiles are organisms that can withstand and even thrive in acidic environments

where the pH values range from 1 to 5 (Gonzalez-Toril, Llobet-Brossa, Casamayor,

Amann & Amils, 2003). The results showed that the P. aeruginosa, B. subtilis, S.

aureus and Salmonella were able to grow in the pH range from 5 to 10 and reached

the maximum growth at pH 7. E. coli was able to grow at pH 5 and reach the

maximum growth at pH 7 but no growth at pH 10.0. These data are in agreement

with that of Yuzo et al who reported that maximum lipase activity from

Pseudomonas fluorescence HU 380 was detected at pH 7 (Yuzo & Sakaya, 2003). The

optimum pH for growing S. aureus in a culture at 37°C is 6.0-7.0. It has a minimum

pH level of 4.0 and a maximum level of 10.0 required for growth. While the optimum

pH for growing Salmonella in a culture at 37°C is 7-7.5. It has a minimum pH level of

3.8 and a maximum level of 9.5 required for growth. The optimum pH for growing E.

coli in a culture at 37°C is 6.0-7.0. It has a minimum pH level of 4.4 and a maximum

level of 9.0 required for growth (Lund, Baird-Parker & Gould, 2000). Bacteria

obtained its nutrients for growth and division from their environment, thus any

change in the concentration of these nutrients would cause a change in the growth

rate. There was a very clear relationship between pH and the size of the inhibition

zone. E. coli appeared to be more tolerant of low pH than of high pH. The reason

why E. coli is not able to tolerate extremely alkaline and acidic environments is

because many of the enzymes that are part of important processes in the E. coli

bacterium are very pH-sensitive. When the change in pH is so extreme, enzymes in E.

coli become denatured, and are prevented from doing their job. Depending on the

enzyme, becoming denatured could cause all sorts of interruptions to biochemical

processes. However, inhibition of enzymes leads to death of the E. coli. Enzymes

typically only have a very small window of tolerance to pH variation, so for the E. coli

to have survived in environments of pH 2.4 - 7.0 is very unlikely (Edick, 1992).

In the fourth experiment we use paraffin oil layer to determine the effect of

oxygen on microorganism growth. From the result, all the microorganisms showed

good growth in the broth without paraffin oil. Exceptionally, P. aeruginosa showed

no growth in the broth with paraffin oil whereas the others microorganisms are

showed moderate growth. This data is in the agreement of Haas et al who reported

that P. aeruginosa is classified as an obligate aerobe (Haas, Gamper & Zimmermann,

1992). Obligate aerobes are totally dependent on oxygen for growth.

Through cellular respiration, these organisms use oxygen to metabolize substances,

like sugars or fats, to obtain energy. In this type of respiration, oxygen serves as the

terminal electron acceptor for the electron transport chain. While the others

microorganisms are classified as facultative anaerobes where can makes ATP by

aerobic respiration if oxygen is present, but is capable of switching to fermentation

or anaerobic if oxygen is absent (Clements, Miller & Streips, 2002).

Conclusion

Environmental factors play an important role in the growth of

microorganisms. Each of these factors is important and may limit growth, it is

generally their combined effects which determine whether microorganism growth

will occur and, give suitable growth condition, which microorganism will grow and

how quickly.

References

Archunan, G. (2004). Microbiology (pp. 267-273). India: Prabhat Kumar Sharma for Sarup & Sons Laser.

Clements, L. D., Miller, B. S., & Streips, U. N. (2002). Comparative Growth Analysis of the Facultative Anaerobes Bacillus subtilis, Bacillus licheniformis, and Escherichia coli. Systematic and Applied Microbiology, 25(2), 284-286. doi: org/10.1078/0723-2020-00108.

Davies, A. R. (1998). Microbiology of meat and poultry (p. 278). United Kingdom, UK: Blackie Academic and Professional.

Edick, G. F. (1992). Escherichia coli: Laboratory Investigations of Protein Biochemistry, Growth and Gene Expression Regulation (3rd ed.). Retrieved from http://www.essaysample.com/essay/001193.html.

Forsythe, S. J. (2000). The Microbiology of Safe Food (p. 26). United State, US: Blackwell Science, Inc.

Gonzalez-Toril, E., Llobet-Brossa, E., Casamayor, E.O., Amann, R., & Amils, R. (2003). Microbial Ecology of an Extreme Acidic Environment, the Tinto River. Applied and Environmental Microbiology, 69: 4853-4865.

Haas, D., Gamper, M., & Zimmermann, A. (1992). Anaerobic control in Pseudomonas aeruginosa (pp. 177–187). In Galli, E., Silver, S., & Witholt, B. (Eds.). Pseudomonas: molecular biology and biotechnology. Washington, D.C.: American Society for Microbiology.

Lund, B.M., Baird-Parker, T.C. & Gould, G. W. (1999). Microbiological Safety and Quality of Food (Vol. 1). United States, US: Aspen Publishers, Inc.

Lund, B. M., Baird-Parker, T. C., & Gould, G. W. (2000). The Microbiology Safety and Quality of Food (Vol. 1). United States, US: Aspen Publisher, Inc.

Paster, T. (2007). The HACCP food safety employee manual. New Jersey. John Wiley& Sons Inc. p17.

Potter, N. N., & Hotchkiss, J. H. (1998). Food science (5th ed., p. 233). United States, US: Aspen Publisher, Inc.

Roday, S. (1999). Food hygiene and sanitation (p. 16). Noida.Tata: McGraw Hill Publishing.

Soni, S. K. (2007). Microbes: A source of energy for 21st century (p. 63). India: New India Publishing Agency.

Srivastava, S. (2003). Understanding bacteria (p. 112). United States,US: Kluwer Academic Publishers.

Todar, K. (2009). Opportunities infections caused by Pseudomonas Aeruginosa. University of Wisconsin-Madison. Retrieved from http://textbookofbacteriology.net/themicrobialworld/Pseudomonas.html

Vanderheijden. (1999). International food safety handbook (p. 33). United States, US: Marcel Dekker Inc.

Yuzo, K., &Sakaya, S. (2003). Purification and characterization of the lipase from Pseudomonas fluorescens HU 380. Journal of Bioscience and Bioengineering, 96, (3), 211-226.