Posted: July 1st, 2015

Pharmaceutical Microbiology

Abstract

The term Z-value is used in the calculation of thermal microbial death time. This is the required temperature for one log 10 D-value reduction. An organism’s D-value is the required time to a given medium, at a temperature that is given, for a reduction of ten-fold in a number of organisms. D-value refers to the reduction time of decimal and is known to be the required time at a specified temperature in order to kill 90 percent of the studied organisms. Z-value is anchored on temperature while D-value is microbiological.

Salmonella ssp. is known to have heat tolerance that is increased in areas where there is low water activity. This is measured through the relative pressure of vapor that is achieved either through solute incorporation or solute drying. Salmonella enteric is one of the species of Salmonella and a member of the family of Enterobacteriaceae. This species is subdivided into subspecies that number to a total of six. These subspecies are salamae (II), enteric (I), indica (VI), arizonae (III a), houtanae (IV) and diarizonae (III b). The habit that is usual for enterica (I) subspecies is of animals that are warm blooded. The rest of the subspecies have a usual habitat of cold-blooded animals and also the environment. (Rhen, 2007)

The purpose of this study is to effectively investigate the activity of low water, the heat resistance of Salmonella enterica serovars in the state of heat resistance in the physiological and humectants type. Serovars are to be placed in low broths before the heat treatment between 48 degrees to 58 degrees for a total time of 30 minutes and samples removed with an interval of 5 minutes difference. The water activity was lowered through the adjustment of its composition in order to contain 20 percent glycerol (aw 0.94), 4 percent NaCl (aw 0.97) or sucrose of 35 percent (aw 0.95).

The humectants type and cell adaptation have a significant effect on the D-value at 55 degrees. The cells that are adapted in 20 percent glycerol had an implication of a low D-value at 55 degrees Celsius. The cells that are not adapted that are placed in broth of 20 percentage glycerol before the heat treatment indicated an increase in the D-value at 55 degrees Celsius in comparison to the adapted cells. More protective effect was seen in NaCl and sucrose under the non-adapted and adapted conditions for the tested serovars when compared to glycerol. In order to fully understand the behavior of the cells in the presence of humectants that are different and the heat response that is subsequent, there is need for more research.

Salmonella serotype has a number of properties associated with it. It has gram-negative non-sporing rods of about 2 to 4 *0.5µm and it doesn’t have capsules. All the salmonella serotypes are known to be potentially pathogenic. However, some are host specific while a majority can affect different hosts. They are usually motile having long flagella. They may create non-motile variants like Salmonella Pullorun and Salmonella Gallinarum. They have optional growth between the temperature rates of 35 degrees to 37 degrees. They can survive freezing, refrigeration and several months away from the host and in much reduced temperatures above 6 degrees to 15 degrees. They are also sensitive to most of the disinfectants and the bacteria are killed in very high temperatures above 60 degrees within 2 to 6 minutes. (Porwollik, 2011)

Heat temperatures have wide effects on the physiological and structural properties of bacteria that are sporulation and non-sporulating. Heat also affects the membrane, ribosomes, RNA, protein, DNA and enzymes. However, there is no single event that can be determined to be responsible for the death of cell. The activation of extracellular alarmones and induction of intracellular heat-shock proteins into vegetable cells exposes lethal temperature that is mildly and vital for cell responses. Several factors contribute to the general resistance to dry or moist heat in bacterial spores. Dry heat induces mutations. The development of heat resistance takes place during sporulation. At this time, there is production of spore-specific heat-shock proteins. There is regaining of heat sensitivity during spores germination.

Methods

In the preparation of test cultures, vials are placed at room temperature. 1.0ml of culture is then transferred TBS of 50ml in flasks of 200ml and then incubated at 37 degrees for 24 hours. There is need to determine the cfu/ml for all the pairs as a control in the flask at zero time. In order to obtain accurate results in the higher temperatures, it is important add the inoculum, mix and sample later in the process. It is also vital to know the viable cells per ml in the Salmonella enterica serovar Typhimurium broth culture in order to get zero time count that is accurate. During the experiment, label eight test tubes that contain 4.5ml of sterile Ringer 10^-1 to 10^-8.

The contexts of Salmonella enterica serovar Typhimurium broth culture is mixed and 500µl of the culture is aseptically removed in the 10^-1 tube and tip discarded then the contents mixed. Using a fresh tip 500µl is removed from the first tube and placed on 10^-2 tube and the process is continuous to the last tubes to 10^-8 dilution. Two TSA plates are marked and inoculated in six equal portions and labeled from 10^-3 to 10^-8. The labels must include sample time, date, name and sample temperature for easy identification and calculation. The process should start with the most dilute and place droplets using sterile Finn tip in each plate at a time until agar is soaked. The viable count on samples should be done at, 10, 20, and 30 minutes after the culture is added and the best method is Miles and Misra method. (Wiedmann & Zhang, 2011)

2ml of culture is the added in the flask with swirling to mix and stop watch should be started immediately. For each sample that is timed requires six test tubes that are labeled from 10^-1 to 10^-6 and each test tube should contain 4.5ml of sterile Ringer. Two sterile TSA plates should be marked in six portions and labeled appropriately in a similar way to the Miles and Misra test plate. The Miles and Misra test procedure is then done in the entire last process.

Results

Below are the results of the data variables in table for class temperature and the graphical results which is then used to determine the D, Z and D₁₂₁ values for the practical work.

Table for Individual pair variable data

Temperature ………OC
Sample Time min	Count one	Count two	Mean count	Dilution used	Cfu/ml original	Log10 cfu/ml
0	12	13	12.5	10-7	2.5107	7.40
5	21	20	20.5	10-5	4.1*105	5.61
10	20	4	12	10-5	2.4*105	5.40
20	3	7	5	10-5	1*105	5.0
30	8	8	8	10-5	1.6*105	5.20

 

 

 

Class data for D at all temperatures

Temperature ˚C	48	52	54	56	58
Number of Observations	9	5	5	5	11
Mean of D	56.739	7.1	5.38	6.4	5.141
Standard Deviation (Std Dev.)	92.5	3.281	2.738	2.035	1.705
Standard Error	30.833	1.467	1.225	0.91	0.514
Mean + Std Error	87.572	8.567	6.605	7.31	5.655
Mean – Std Error	25.906	5.633	4.155	5.49	4.627
Log ₁₀ of Mean of D	1.754	0.851	0.731	0.806	0.711
Log ₁₀ Mean + Std Error	32.587	2.318	1.956	1.716	1.225
Log ₁₀ Mean – Std Error	-29.079	-0.616	-0.494	-0.104	0.197

 

 

 

 

 

 

 

 

Calculating value of Z:

Z value = difference in temperatures within 1 log which is the D value.

= 58^oC – 48^oC

= 10^oC

Correct calculation of D₁₂₁

D₁₂₁= 90 /100 * 10

= 9 minutes

Discussion

 

 

Reference

Acton, A. (2013). Advances in Salmonella enterica Research and Application. Atlanta: Scholarly

Editions.

Porwollik, S. (2011). Salmonella: From Genome to Function. Norfolk: Caister Academic Press.

Rhen, M. (2007). Salmonella: Molecular Biology and Pathogenesis. Wymondham: Horizon

Biosceince.

Wiedmann, M., & Zhang, W. (2011). Genomics of Foodborne Bacterial Pathogens. New York:

Springer Publishing Press.