FE-204 exp.3

Temperature Lethal Effect and Membrane Filtration Method

Purpose

Behelding of the temperature lethal effect to microorganisms and calculation of the number of the microorganism in tap water by using membrane filtration method.

Theory

Temperature Lethal Effects

Temperature’s lethal effects on bacterial growth can be seen after subjecting various bacterial cultures to excessive heat in the range of 90 to 100 degrees C. Microorganisms are killed by elevated temperatures mainly because of the susceptibility of their macromolecules to heat. High temperatures cause proteins to denature and unfold which results in the loss of the tertiary structure and loss of overall function. Most proteins are enzymes which means that metabolic activity relies on their function. If these proteins are damaged the metabolic capabilities of the organism will be irreversibly damaged. Nucleic acids are also damaged by heat resulting in the loss of structure of DNA and RNA. This will lead directly to cell death. Smaller co-factors like NAD+ can also be damaged by heat and loss of these factors leads to cell damage and death. Some cells like that of Bacillus and Clostridium are more resistant to heat because of their endospores. These endospores contain calcium dipicolinate that protects the cells from heat. Endospore-specific proteins can bind to nucleic acids and prevent denaturation.

The Membrane Filtration (MF) method is a fast, simple way to estimate bacterial populations in water that is low in turbidity. This method is especially useful when evaluating large sample volumes or performing many tests daily.

In the initial step, an appropriate sample volume passes through a membrane filter with a pore size small enough (0.45 micron) to retain the bacteria present. The filter is placed on an absorbent pad (in a petri dish) saturated with a culture medium that is selective for the growth of a specific organism. The petri dish containing the filter and pad is incubated, inverted, for 24 hours at the appropriate temperature. After incubation, the colonies that have grown are identified and counted using a low-power microscope.

Materials

Bunsen burner

E.coli

Glass spreader

Petri plates

Water both

Pipette

Match

Filter paper

Incubator

Funnel

Coarse Filter

Membrane filter

Sticker

Forcep

Tap water

Procedure

E.coli broth was taken and it was exposed to heat at water both each temperature (60C and 70C) and was worked different  time interval 0,5,10,15 minutes.After ….

Results

Discussion

The High temperatures have profound effects on the structural and physiological properties of sporulating and non-sporulating bacteria, with membranes, RNA, DNA, ribosomes, protein and enzymes all affected. Nevertheless, it is apparent that no one single event is responsible for cell death. The induction of intracellular heat-shock proteins and the activation of extracellular alarmones in vegetative cells exposed to mildly lethal temperatures are important cell responses. In bacterial spores, several factors contribute to the overall resistance to moist (wet) and dry heat; the latter, but not the former, induces mutations, Heat resistance develops during sporulation, when spore-specific heat-shock proteins are also produced. Heat sensitivity is regained during germination of spores.

In experiment we obtention some values which base different times (0,5,10,15 minutes) with temperatures (60,70) to determine the TDP( Thermal death point) and TDT (Thermal death time) for sample E.coli broth culture. TDP was determined as 60C but in that value we observed some microorganism.Maybe unwanted contaminations occurred.The other values are average of the results which determined by groups.

The Membrane Filter Technique is an effective, accepted technique for testing fluid samples for microbiological contamination. It involves less preparation than many traditional methods, and is one of a few methods that will allow the isolation and enumeration of microorganisms. The MF Technique also provides presence or absence information within 24 hours.

In the membrane filtration method, the concentration of larger samples on a membrane filter is a key benefit of the technique over the Most Proable Number (MPN) procedure as well as over pour plate and spread plate techniques.