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Yuxuan Chang 11199771

August 1, 2013

Project 5: Investigation of the effect of heat on bacteria using plate count methods
Aim To investigate the effect of heat on gram positive and gram negative bacteria by serial dilutions and plate count methods.

Introduction
Heat treatment is the most widely used and effective sterilization method in food industry. In general, the removal of microorganisms is achieved by the denaturation of proteins and enzymes through heating at high temperature. This method can be further divided into moist heat sterilization and dry heat sterilization. The major difference is come from the heating media adopted. Steam is used in moist sterilization process while heated dry air or fire is used in dry heat sterilization (Prince, 2012). In practice, moist heat sterilization is a better choice for heat sensitive and steam permeable material such as culture media and aqueous solution (Kumar, 2011). Steam impermeable materials including powders and glassware are sterilized by dry heat. Temperature and exposure time are two important parameters in heat sterilization. Thermal death times (TDT) and D value define the time required at certain temperature to kill 100% and 90% of the bacteria respectively. This experiment was focused on the thermal death times (TDT) and decimal reduction times (D value) of Bacillus subtilis at 65C. The viable organism count was accomplished through serial dilution and plating on nutrient agar.

Procedure

Refer to 280.201/162.214 Industrial Microbiology/Biology of Microorganisms Laboratory Manual for procedure details.

Microbiological media
Table1. Media summary Name
Molten Nutrient Agar 52C Sterile Peptone Water

Final pH
7.40.2 7.20.2

Sterilizing process
Sterilize by autoclaving at 121C for 15 minutes Sterilize by autoclaving at 121C for 15 minutes. (Bridson, 1998)

Purpose
For the multiplication of bacteria For serial dilutions

Results and Discussion The number of colonies on each plate was summarized in table 2.
Dilution 0 mins 5 mins 10 mins 15 mins 20 mins 30 mins 10^(-1) TNTC TNTC TNTC TNTC TNTC TNTC 10^(-2) TNTC 720 459 TNTC 528 337 10^(-3) TNTC 42 72 63 102 TNTC 10^(-4) TNTC 9 7 8 8 9 10^(-5) 82 0 0 1 0 1 10^(-6) 6 0 4 1 0 0

Table 2. Growth of each organism on 4 Agar plates

Yuxuan Chang 11199771


August 1, 2013

Note that TNTC means too numerous to count.

The survivor curves were plotted from the data shown in table 2. The points whose colony number are TNTC and 0 were not included in the graph.
7.5 7 6.5 6 5.5 5 4.5 4 3.5 0 5 10 15 y = -0.1343x + 5.9862 20 25 30 35 10^(-2) 10^(-3) 10^(-4) 10^(-5) 10^(-6)

log number

Time/min

Figure 1. log10 number of surviving cells vs time of B.Subtilis at 65C. Despite the dilution of 10-4, a general trend of decreasing colony numbers with time was observed. However, from the dilutions of 10-4, the number of the colonies increased. Therefore the effect of heat on B. Subtilis could not be concluded accurately due to the inconsistence of results. The TDT value of the strain could not be calculated as the bacteria were not completely killed in most of the diluted samples at 30mins. It is recommended that longer time or higher temperature is required to achieve 100% killing. The D value was determined from the dilution of 10-6, which was 7.4 min the

Conclusion
Reference list Bridson, E. Y. The Oxoid Manual. Basingstoke, Hampshire, England: Unipath, 1998. Print. Lal, A.,& Cheeptham,N. (2007). Eosin-Methylene Blue Agar Plates Protocol. Retrieved from http://www.microbelibrary.org/component/resource/laboratory-test/2869-eosinmethylene-blue-Agar-plates-protocol. Power, D.A. Zimbro,M.J., & Miller,S.M (2009). Difco& BBL Manual Manual of Microbiological Culture Media. United States: Sparks, MD : Becton Dickinson and Co. http://www.ehow.com/about_5418872_effect-temperature-bacterial-growth.html http://www.pharmainfo.net/siriki-praveen-kumar/blog/principles-sterilization http://www.gibraltarlabsinc.com/gibraltarblog/difference-between-moist-heatsterilization-dry-heat-sterilization/ (prince)

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