Effect of temperature on growth of microorganisms- TDT and TDP

 Aim

To determine the thermal death time and thermal death point of the given test organism

Principle

Temperature is one of the most important physical factors influencing the growth of microorganisms. Bacteria unlike eukaryotes lack homeostatic mechanism and they do not regulate the heat generated by metabolism thus are affected readily by changes in temperature. Enzymatic reactions have maximum efficiency at optimum temperature which varies with organisms. For every 10⁰C rise in temperature, there is 2 fold increase in the rate of enzyme catalyzed reactions for a limited range of temperature. At high temperatures, proteins are irreversibly denatured and there is a total enzyme destruction. At low temperature, the enzyme reactions are merely inactivated and are thus less harmful.

Bacteria are divided into three major groups with respect to their temperature requirements:

1)     Psychrophiles with optimum temperature between 0 and 20⁰C

2)     Mesophiles with optimum temperature between 20 and 40⁰C

3)     Thermophiles with optimum temperature between 40 and 60⁰C

Normally, the lethal range of temperature for bacteria is between 50 and 100⁰C. Time of exposure is a vital factor in assessing the lethal effect of high temperature on bacterial cells. Determination of thermal death time (TDT) and thermal death point (TDP) are done for this purpose.

1)     Thermal death point (TDP) – Temperature at which an organism is killed in 10 minutes of exposure. Lethal action of heat has a temperature-time relationship. Thermal death point is done to determine the degree of heat tolerance of the organism. Some factors such as pH, moisture, composition of media and age of cells influence TDP.

     2)     Thermal death time (TDT) – The time required to kill cells/spores at a given temperature. The length of time that the microbes are exposed to heat contributes to lethal effect. This is assessed by exposing cells to fixed temperature which is determined as thermal death time for increasing periods of time.

 

Materials required

Culture of  E. coli, nutrient tubes, nutrient agar plates, water bath, incubator etc

Methodology

1)     Thermal death point (TDP)

 Into each of the sterile test tubes, 5 ml of sterile nutrient broth was dispensed and tubes were marked 40, 50, 60, 70, 80, 90 and 100 ⁰C for different organisms. A loopful of culture was inoculated into the respective tubes and incubated for ten minutes at each temperature. They were then plated on nutrient agar plates and incubated at 37⁰C for 24 hours. The temperature above which the organism was completely killed and did not grow was noted and this was determined as the thermal death point of the organism.

2)     Thermal death time (TDT)

Each sterile tube containing 5 ml nutrient broth was inoculated with loopful of cultures and incubated at their thermal death point (TDP). A loopful of cultures from the tubes were taken at regular intervals of 3 minutes each, starting from 0 minutes till 15 minutes and plated on to nutrient agar followed by incubation at  37⁰C for 24 hours. The time period above which the organisms were completely killed and did not grow was determined as thermal death time (TDT).

Result

The thermal death time (TDT)  and thermal death point (TDP) for E. coli was 15 minutes at 80⁰C.

Effect of aeration on growth of bacteria

 Aim

To study the effect of aeration on growth of bacteria

Principle

Microorganisms are classified into categories such as aerobic, facultative anaerobic, and anaerobic bacteria depending on their metabolic strategies influenced by the availability of oxygen. Aerobic bacteria or obligate aerobes, require oxygen for their growth and survival as they utilize oxygen as the final electron acceptor in their electron transport chain that yields a large amount of ATP. In contrast, anaerobic bacteria or obligate anaerobes do not require oxygen for growth and they rely on anaerobic respiration or fermentation, metabolic pathways that generate energy without oxygen by using alternative electron acceptors such as nitrate, sulfate, carbon dioxide or by relying solely on substrate-level phosphorylation. Facultative anaerobic bacteria possess metabolic flexibility to grow both in the presence or absence of oxygen.

Aeration is a critical factor in microbial growth, particularly for aerobic and facultative anaerobic microorganisms, as it directly impacts the availability of dissolved oxygen (DO) in the culture medium. Oxygen is essential for respiration and other metabolic processes in aerobic organisms, serving as the final electron acceptor in the electron transport chain.  For facultative anaerobic bacteria, even though they can survive and grow both with or without oxygen, aeration plays a significant role in their growth.  When oxygen is available, they preferentially switch to aerobic respiration since it is metabolically much more efficient. As a result, in well-aerated conditions, aerobes and facultative anaerobes will exhibit faster growth rates, reach higher cell densities, and utilize their carbon sources more completely than in unaerated conditions.

Materials required

1. Culture of E coli and Bacillus spp.

2. Sterile Nutrient broth flasks, Shaker incubator, other routine microbiological facilities

 

Procedure

1. Nutrient broth flasks were labelled with the organism to be inoculated, E coli and Bacillus spp.

2. One set of inoculated flasks was placed on an orbital shaker incubator set to 37°C with a shaking speed of 150-250 rpm. A second set of inoculated flasks was placed in a static incubator at 37°C. Both were incubated for 24-48 hours.

3. The flasks were observed, after incubation and the optical density at 600 nm was recorded

 

Observation and result

The bacterial species were found to have different growth patterns under aerated and static conditions.  For both organisms, maximum growth was observed under aerated conditions.

 

  

Effect of aeration on bacterial growth (left hand side)

Microorganism	OD at 600 nm
	Under static conditions	At shaker incubator 150 rpm
E coli	0.24	0.90
Bacillus sp	0.11	0.58

 

 

 

Effect of temperature on growth of microorganisms- TDT and TDP

 Aim

To determine the thermal death time and thermal death point of the given test organism

Principle

Temperature is one of the most important physical factors influencing the growth of microorganisms. Bacteria unlike eukaryotes lack homeostatic mechanism and they do not regulate the heat generated by metabolism thus are affected readily by changes in temperature. Enzymatic reactions have maximum efficiency at optimum temperature which varies with organisms. For every 10⁰C rise in temperature, there is 2 fold increase in the rate of enzyme catalyzed reactions for a limited range of temperature. At high temperatures, proteins are irreversibly denatured and there is a total enzyme destruction. At low temperature, the enzyme reactions are merely inactivated and are thus less harmful.

Bacteria are divided into three major groups with respect to their temperature requirements:

1)     Psychrophiles with optimum temperature between 0 and 20⁰C

2)     Mesophiles with optimum temperature between 20 and 40⁰C

3)     Thermophiles with optimum temperature between 40 and 60⁰C

Normally, the lethal range of temperature for bacteria is between 50 and 100⁰C. Time of exposure is a vital factor in assessing the lethal effect of high temperature on bacterial cells. Determination of thermal death time (TDT) and thermal death point (TDP) are done for this purpose.

1)     Thermal death point (TDP) – Temperature at which an organism is killed in 10 minutes of exposure. Lethal action of heat has a temperature-time relationship. Thermal death point is done to determine the degree of heat tolerance of the organism. Some factors such as pH, moisture, composition of media and age of cells influence TDP.

     2)     Thermal death time (TDT) – The time required to kill cells/spores at a given temperature. The length of time that the microbes are exposed to heat contributes to lethal effect. This is assessed by exposing cells to fixed temperature which is determined as thermal death time for increasing periods of time.

 

Materials required

Cultures of S. aureus, E. coli, nutrient tubes, nutrient agar plates, water bath, incubator etc

Methodology

1)     Thermal death point (TDP)

 Into each of the sterile test tubes, 5 ml of sterile nutrient broth was dispensed and tubes were marked 40, 50, 60, 70, 80, 90 and 100 ⁰C for different organisms. A loopful of culture was inoculated into the respective tubes and incubated for ten minutes at each temperature. They were then plated on nutrient agar plates and incubated at 37⁰C for 24 hours. The temperature above which the organism was completely killed and did not grow was noted and this was determined as the thermal death point of the organism.

2)     Thermal death time (TDT)

Each sterile tube containing 5 ml nutrient broth was inoculated with loopful of cultures and incubated at their thermal death point (TDP). A loopful of cultures from the tubes were taken at regular intervals of 3 minutes each, starting from 0 minutes till 15 minutes and plated on to nutrient agar followed by incubation at  37⁰C for 24 hours. The time period above which the organisms were completely killed and did not grow was determined as thermal death time (TDT).

Result

The thermal death point (TDP) and thermal death time (TDT) for S. aureus was --------- and for E. coli was ----------.

Air sampling by open plate method

 Aim 

To compare air flora of various locations by plate exposure technique. 

Principle 

Air is not a natural environment for the growth and reproduction of microorganisms. It doesn’t contain the necessary amount of moisture and utilizable form of nutrients. Air is mainly a transport and/or dispersal medium for microorganisms which occur in relatively small numbers in air when compared with soil or water. Microorganisms are still found in air and their presence is of considerable importance economically and to public health. 

Microorganisms in the air include mainly vegetative cells and spores of bacteria, fungi and algae, viruses and protozoan cysts. The species vary depending on the locality. However, certain forms are quite uniformly present. Molds and yeasts are commonly present in the air and in some localities, they outnumber bacteria. They produce spores which are capable of resisting unfavourable conditions for long periods of time. The air microflora consists of molds such as Aspergillus, Penicillium, Alternaria, their spores etc. and of bacteria including spore forming and non-spore forming Gram positive bacilli, Gram positive cocci and Gram negative rods. The aerobic spore forming Bacilli from soil are quite frequently seen in air. Bacillus subtilis, known as Hay Bacillus, is one such common bacteria. Microorganisms are introduced into the air by various sources, most important being the dust particles which contain vegetative cells and spores. 

The microbiological quality of air is dependent upon factors such as temperature, relative humidity, amount of dust stirred up and exposure to ultraviolet or electromagnetic radiation. 

Air sampling is undertaken in areas such as operation theatres, pharmacy sterile units and sterile supply units to assess air quality. It may be used as a continuous monitoring system e.g. in laboratories, in order to assess fluctuations in background counts which may contaminate cultures. Sampling may be either active or passive. Active sampling involves the use of mechanical equipment which draws in known volumes of air which then impinge on culture media or filters. Numbers of microbes present per unit volume of air can then be calculated accurately. Passive sampling needs no special equipment – agar plates containing a suitable medium are horizontally exposed in the area for a defined period of time. The plates are then incubated at a suitable temperature and the number of colonies which develop are then counted. Several plates would usually be exposed at the same time in order to assess the average microbial count. This method is time-consuming and needs careful interpretation as air movements and activity may lead to wide fluctuations in results. 

Settle-plate technique has been widely used to study the air quality outdoor and indoor. The number of organisms present in the air at a given time is dependent upon the activity in the environment and the amount of dust stirred up. An active environment shows a higher microbial count than an inactive one. The numbers in a dusty, untidy room are greater than in a clean room. 

Materials Required 

Media - Nutrient Agar and Sabouraud’s Dextrose agar 

Equipment - Bunsen burner, Petri dishes, incubator 

Procedure 

1. Nutrient agar and Sabouraud’s Dextrose agar were prepared and poured into petri dishes 

2. The petriplates were exposed to air in flow, at different locations. In each location, two petriplates with medium were exposed, for different time intervals such as 5 and 10 minutes. 

3. The petriplates were then closed and nutrient agar plates incubated at 37^oC for 24 hours and Sabouraud’s Dextrose agar plates at room temperature for 3-5 days. 

4. After incubation, the plates were observed and colony count noted. 

Observation and Result 

The number of colonies developed in Nutrient Agar and Sabouraud’s Dextrose agar plates exposed at various locations were determined

                Air sampling by plate exposure method (Left hand side) 

	Number of colonies on Nutrient agar plate
Location	After 5 minutes exposure	After 10 minutes exposure
Lab	14	20
Library	12	29
Laminar air flow chamber	4	10
Outdoor	27	40

 

 

 

 

 

 

	Number of colonies on SDA plate
Location	After 5 minutes exposure	After 10 minutes exposure
Lab	10	16
Library	11	21
Laminar air flow chamber	5	8
Outdoor	18	31

Cultivation of fungi by slide culture technique

 Aim

To observe morphological characteristics of Fungus

 

Principle

Fungi comprises both molds and yeasts. Molds are filamentous and multi cellular whereas yeasts are usually unicellular.  Fungi possess rigid cell walls containing chitin, mannan and other polysaccharides. They possess true nuclei.  Fungi are important in both beneficial and harmful ways. They act as decomposers, certain fungi are parasites, cause diseases in plants, humans and other animals, they are important in many industrial processes including the making of bread, wine, organic acids, antibiotics, etc. and are important research tools. 

    Morphologically, a mold or filamentous fungi consists of long, branched, threadlike filaments of cells called hyphae that form a mycelium, a tangled mass like aggregation.  

    The isolation, culture, and microscopic examination of molds require the use of suitable selective media and special microscopic slide techniques. The process of transferring hyphae to a slide for staining and observation will break up the structure of hyphae and sporangiophores, so the identification will be very difficult. This problem could be resolved by the slide culture technique where, sporangiophores, hyphae and spores remain intact during staining.

 

Materials required

Cultures - culture of Penicillium and Aspergillus

Reagents - lactophenol cotton blue stain, Ethyl alcohol

Equipments - Bunsen burner, inoculating needle, staining tray, microscope, sterile Petri dish, Filter paper, triangle shaped glass rod, Microscope slides, coverslips, Inoculating needle, forceps, etc.

 

Procedure

1. A triangle shaped glass rod, glass slide, cover slip and a moist piece of cotton was placed in a Petri dish and the set up was sterilized.

2. Using a flame sterilized scalpel, 5 mm square block of Sabouraud’s dextrose medium was cut from the plate of Sabouraud’s agar.

3. The block of agar was aseptically transferred to the slide.

4. The four sides of the agar square was inoculated with the fungus and the coverslip was placed on the upper surface of the inoculated agar block.

5. The set up was covered using the petri dish lid and kept at room temperature for 48 hours.

6. After incubation, the coverslip was carefully removed, the agar block separated from the glass and the coverslip and glass slide were stained separately using lactophenol cotton blue and observed under microscope.

 

Observation

The fungal growth in the first slide culture plate appeared first white and then blackish with a pale colour on the reverse side and colonies in the second slide culture plate appeared greenish blue in colour with a wrinkled appearance on the reverse side. 

 

 

 

Result

On microscopic observation, organisms from the first slide culture plate was identified as Aspergillus species and that from the second slide culture plate was identified as Penicillium species.

 

                                              

Fungal Staining

      Aim

To perform fungal staining and to observe fungi 

       Principle

The lactophenol cotton blue (LPCB) wet mount is most widely used method for the staining and observation of fungi.  The LPCB stain has following three components, Phenol to kill any live organism, Lactic acid to preserve fungal structures and Cotton blue to stain the chitin and cellulose of the fungal cell wall in blue colour. 

       Materials required

1. Cultures – Cultures of Penicillium, Aspergillus

2. Reagents – lactophenol cotton blue

3. Equipments – Glass slides, cover slips, Bunsen burner, inoculating needle, staining tray, microscope

 

           Procedure

1. A drop of lacto phenol cotton blue was put on a clean microscopic glass slide
2. A portion of mycelial mat from the fungal culture was transferred in the drop of lactophenol cotton blue using a flamed and cooled inoculation needle.
3. The fungal mycelia were gently spread and mixed with the stain using the inoculation needle. 
4. A cover slip was kept on the surface of slide and observed under microscope.              

Observation 

The slide was observed under low and high power objectives of the microscope and type of spores and arrangement of hyphae noted.

Result

Aspergillus spp. is  recognized by its conidiophores terminating in an apical vesicle and phialides are attached to the vesicle and bear conidia in chains. 

Penicillium spp. is identified by conidiophores with secondary branches that give it a brush-like appearance. Spores (conidia) are produced in chains from the tips of the phialides.

                                                                      Aspergillus

                                                                             Penicillium