Etiket Arşivleri: microorganisms

Pure Culture Techniques ( Jackie Reynolds )


Most specimens (from animal tissue, plant tissue, or environmental samples) will be mixed, with a variety of bacteria (or other microorganisms). A single gram of feces, for example, has over 1010 bacteria and that gram would have over 20 different bacterial species.
Particularly in a medical setting, where a patient’s body fluid or tissue (skin, blood, spinal fluid, urine) is sent to the microbiology lab for analysis, the specimens will most often be mixed. In order to identify the bacteria and run antibiotic susceptibilities on the causative agent of the infection, the microbiologist must be working with a pure culture of the bacterium. This exercise begins with a mixed culture of bacteria and will end with, hopefully, pure cultures of the 2 bacteria. Luckily, the 2 bacteria being used look different from each other when growing on agar plates. Two different types of agar plate isolation techniques will be used—streak and pour. Each method has advantages and disadvantages, and particular uses. The same mixed culture will be used for both methods. Another technique, called the
spread plate, is used commonly for counts, as is the pour plate technique. It uses pre-made agar plates, with the fluid inoculum being placed on top of the agar medium. The inoculum is then spread around on the plate with a bent glass rod. That procedure will not be done today. This also gives you a chance to see how agar plates are made. Some of the TSA plates are already made: others will be made by you. This requires that liquefied agar medium (sitting
in a water bath) be kept above the solidification temperature. Agar’s solidification temperature is 42 degrees C, but its liquefaction temperature is 100 degrees C. When sterilized in an autoclave or boiled, the medium will stay liquid until the temperature gets to 42 C. When that happens, the medium will solidify very fast. If it solidifies on you before you finish making your cultures, it has to be disposed of (if not used yet, it can be placed back in the sterile media racks).


ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate in the autoclave bag. If you see water running on the agar plate, you can do 2 things:
 Place the agar plate upside down in the 37C incubator with the top cracked.
 Get another agar plate.


Laboratory‎ > ‎Effect of Temperature on Growth of Microorganisms & Osmotic Pressure



FATİH KOCABAŞ 09/12/2005
1274661 GENE Friday
Group : 07


To know how to control bacterial growth physically
To be able to learn that each organism have an optimum temperature at which growth rate at the highest level
To be able to recognize that temperature has a big affect on bacterial enzymes
To be able to determine thermal death point and thermal death time for microorganisms
To be able to explain heat-resistance differences between selected bacterial cultures
To be able to recognize that high-solute concentration has an affect on bacterial growth.
Observe the effect of NaCl on bacterial growth
To be able to distinguish the bacterial species with respect to their salt tolerances


Temperature effects on growth
1. Label the 4 N.B tube by writing species that used, incubation temperature, group and section.
2. By using aseptic techniques inoculate Escherichia coli and Serratia marcescens from N.B stock culture by the use of inoculating loop. For our group numbr 7 we used E. coli and incubated at 37°C.
3. Incubate each at different incubation temperatures: 5°C, 25°C, 37°C, 42°C for 24 hours incubation time.
4. Observe the growth and turbidity (read the absorbance of the NB of the tubes) by the use of spectrophotometer at OD: 595nm.
5. Concentration indicates the bacterial density
6. We used control NB, so that the absorbance of NB does not affect bacterial absorbance. NB used for calibrations
Secondly, label the two slants with section, group #, bacteria name (E. coli & S. marcescens). Aseptically inoculate the bacteria using sterilized loop to agar slants, then incubate at 50C, 250C and 370C o/n. After one week we observed the growth (color of the growth).

Temperature lethal effects: for group 7

• Label the 5 plate by writing species that used, exposure time (control, 10’, 20’, 30’, 40’ and 50’), group number and section.
• We used Bacillus subtilis in group7 at 90 Co for zero, 10, and 20 minutes
• By using aseptic techniques, inoculate 0.1 ml Bacillus subtilis from N.B culture on the agar surface by using spread plate technique as a control plate.
• Adjust the temperature by using water bath at 90°C
• Put the culture into he water bath with nonsterile N.B tube and put the thermometer into the nonsterile N.B culture to have the culture temperature correctly without contamination.
• After 10 min. At 90°C, by using aseptic techniques, inoculate 0.1 ml Bacillus subtilis from N.B culture on the agar surface by using spread plate technique
• Until the last 50 min. Repeat the inoculation process after each 10 min. waiting of the culture into the 90°C water bath.
• Incubate at 37°C for 24 hours.

Osmotic pressure & bacterial growth:

• Label previously prepared 5 plates containing 0, 0.5, 5, 10, 15 M % NaCl plates by writing group number, section and species names as below

• By using aceptic techniques, Make the inoculation of each species under its name as a single line by using a needle
• After one week, we observed the growth and compared results.
Results & Observations

Table (Absorbance values of NB cultures at OD595):
Temperature (Co) Absorbance (E.coli) Absorbance (S.marcescens)
5 0 0
25 0,543 1,037
37 0,869 1,357
50 0 0,028

Salt Agar Plates
Species/Salt C 0 % NaCl 0.5 % NaCl 5% NaCl 10 % NaCl 15 % NaCl
E. coli +++ +++ + – –
S. aureus +++ +++ ++ + –
B. subtilis +++ +++ + – –

Agar Slants:
Species/Temperature 5oC 25oC 37oC
E. coli 0 (No growth) + + white colonies/no pigment formation
S. marcescens 0 (No growth) + red/pink + white colonies/no pigment formation

Temperature effect table:
45 °C 0 min 10 min 20 min 30 min 40 min 50 min
E. coli uc uc uc uc uc uc
B. subtilis uc uc uc uc uc uc
60 °C
E. coli uc uc 432 24 1* 64*
B. subtilis uc uc uc 112 17 1
75 °C
E. coli uc 2 3 0 0 0
B. subtilis uc 0 1 0 0 0
90 °C
E. coli uc 0 0 0 0 0
B. subtilis uc 0 0 ?* 7 1


In this experiment our purpose was that to observe the temperature effects on growth to found the values of TDP( thermal death point) and TDT(thermal death temperature) and lastly ,to observe the effect of osmotic pressure on bacterial growth.

To observe the effect of temperature on bacterial growth, we make the E. coli incubation at different temperatures; 5 °C, 25 °C, and for our group 37°C. After 24 hour incubation time we observed the growth not by spectrophotometer measurement at 595nm but just looking at the turbidity and absorbance values. As result of the observation we had an expected result that, the biggest growth takes place at around 37°C ranging from 34 t0 38°C. We were expected to see that 37°C is the optimum temperature (the temperature at which the biggest growth takes place) for E. coli but from the graph given in results part seems like more likely 35°C. E. coli is a bacterium living in the human body also and it is clear that the optimum temperature of it will be about the body temperature 37°C. S.marcescens also grows at around 37°C well. These prove that these two bacteria are mesophilic.

Another aim of the experiment was to determine the TDP & TDT. In order to do that, we made the inoculation at constant temperature in every 10 min. In the experiment we used E. coli at 90°C from 0 to 20 min. As indicated in the result chart we could determine the TDP & TDT. According to the data for E. coli TDP (thermal death point; lowest temperature required to kill all microbes in liquid suspension in ten minutes) is near to 75°C and I think it might be 80°C. However, when we come to B. subtilis, TDP is exactly 75°C from our experiment. To determine the TDT (thermal death time; minimal time required to kill all microorganisms keeping temperature constant) we should consider three different temperatures 60°C, 75°C , 90°C. For example, for E. coli at constant 60°C, we could conclude if we ignore error and accept it like no growth I can say that at 60°C TDT is 40min, for 75°C; 30min and for 90 (our group); 10 min is enough to kill all.

To observe the effects of osmotic pressure on bacterial growth we use agar plates that contain different concentration of NaCl, and then we can observe the salt tolerance of the microorganisms. We use 0.5% NaCl and see that the biggest growth is shown by S. aureus and then others come. We cannot distinguish difference between E. coli and B. subtilis but no one can tolerate 15 % NaCl of high concentration of salt. It is clear that all species can tolerate 0.5% NaCl concentration. Finally, we should note that S. aureus is very salt tolerant. I should emphasize that E. coli is a halophilic bacteria, but in our case it is a mild halophile. In addition, B. subtilis nearly give the same reaction as E. coli to salt concentrations. But S.aureus seems to be more hypotolerant. We know that E.coli and S.aureus can live in our body. And the experimental results prove this and correlate with our average body salt concentration 0.85% and these two bacteria can grow well at this salt concentration.

Finally, when we come to slant agars, in our experiment we observed that neither Serratia marcescens nor Escherichia Coli can grow at 5°C but both can grow at 25°C and 37°C. Moreover, S. marcescens produce pigments at 250C however it is colorless at 370C. The pigment in S. marcescens is called prodigiosin. Once the growth temperature of S. marcescens is raised to 370C, we know that the pigment stop being produced. It is believed that an enzyme used in the production of prodigiosin is affected by temperature so that pigment is no longer made. However E.coli never forms pigment.


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