Eportfolio week 13

During the lectures, we have a quiz section where each groups prepared some questions. There are some questions asked such as what will happen if during the smear preparation if the specimen is overheated during heat fixation, the definition of heat fixation, what are different types of hemolysis and the reagents used for gram-staining. There are questions asked on what is acid-fast staining and which types of organisms suitable to grow in 7.5 % NaCl nutrient agar plates. Group 7 answered the most questions and was the winner of the quiz .

Eportfolio week 12

Experiment 18: Serial Dilution-Agar Plate Procedure to Quantitate Viable Cells
From this experiment, I learnt how to enumerate the microorganisms in a given sample. There are many ways to determine the microbial growth. There are direct counting methods, chemical methods, measure the tubidity and the serial-dilution plate method. In this experiment, we do the serial-dilution plate method. From the given sample of E. coli, we carrries out a series of dilution. Then, we dilute the sample further by using pour plate and spread plate to get single colonies. After incubation, we count the single colonies present by using the Quebec colony counter. The number of colonies counted are taken for a range between 30 too 300. The number of colonies which is over 300 are labelled as TNTC while TFTC is labelled for plate which has colonies of less that 30.The procedures of the experiment are as followed. The procedures of the experiment are as followed. Six agar deep tubes are liquefied in an autoclave or by boiling. The molten agar tubes was cooled and maintained in a water bath at 45 °C . The E. coli culture tube are labelled with the number 1 and the seven 9-ml water blanks as numbers 2 through 8. The labelled tubes were placed in a test tube rack. The Petri dishes are labelled as 1A, 1B, 2A, 2B, 3A, and 3B. The E. coli culture was mixed by rolling the tube between the palms of the hands to ensure even dispersal of cells in the culture. Then, 1 ml from the bacterial suspension, Tube 1, was aseptically transferred to water blank Tube 2 with a sterile pipette. The pipette was discarded in the beaker of disinfectant. The culture has been diluted 10 times to 10^-1. Tube 2 was mixed. With a fresh pipette, 1 ml from Tube 2 was transferred to Tube 3. The pipette was discarded. The culture has been diluted 100 times to 10^-2. Tube 3 was mixed. With a fresh pipette, 1 ml from Tube 3 was transferred to Tube 4. The pipette was discarded. The culture has been diluted 1000 times to 10^-3.  Tube 4 was mixed. With a fresh pipette, 1 ml from Tube 4 was transferred to Tube 5. The pipette was discarded. The culture has been diluted 10000 times to 10^-4.  Tube 5 was mixed. With a fresh pipette, 0.1 ml from Tube 5 was transferred to Plate 1A. The pipette was returned to Tube 5 and 1 ml from Tube 5 is transferred to Tube 6. The pipette was discarded. The culture has been diluted 100000 times to 10^-5.  Tube 6 was mixed. With a fresh pipette, 1 ml of this suspension from Tube 6 was transferred to Plate 1B. The pipette was returned to Tube 6 and 0.1 ml was transferred from Tube 6 to Plate 2A. The pipette is returned to Tube 6 and1 ml from Tube 6 is transferred to Tube 7. The pipette was discarded. The culture has been diluted 1000000 times to 10^-6.  Tube 7 was mixed. With a fresh pipette, 1 ml of this suspension from Tube 7 was transferred to Plate 2B. The pipette was returned to Tube 7 and 0.1 ml was transferred from Tube 7 to Plate 3A. The pipette is returned to Tube 7 and1 ml from Tube 7 is transferred to Tube 8. The pipette was discarded. The culture has been diluted 10000000 times to 10^-7. Tube 8 was mixed. 1 ml of this suspension was transferred from Tube 8 to Plate 3B with a fresh pipette. The pipette was discarded. The dilute procedure is now complete.

            The temperature of the molten agar medium was checked to be sure the temperature is 45 °C. A tube was removed from the water bath and the outside surface was dried with a paper towel. The agar is pour into Plate 1A by using the pour-plate technique. The plate was rotated gently to ensure uniform distribution of the cells in the medium. This step is repeated for the addition of molten nutrient agar to Plates 1B, 2A, 2B, 3A, and 3B. Once the agar solidified, the plates is incubated in an inverted position for 24 hours at 37 °C.

The spread plate was also carried out to get single colony. Bacterial suspensions are prepared as described above and agar plates were labelled accordingly. The bent glass rod was placed into a beaker and a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. An appropriately labelled nutrient agar was placed on the turntable. 0.1 ml of bacterial suspension was placed on the center of the plate with a sterile pipette. The glass rod was removed from the beaker and it was passed through the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the burning alcohol from running down the arm. The alcohol was allowed to burn off the rod completely. The rod was cooled for 10 to 15 seconds. The Petri dish cover was removed. In the absence of a turntable, the Petri dish was turned manually and the culture was spreaded with the sterile bent glass rod.

Using a Quabec colony counter and a mechanical hand counter, all colonies were observed on plates. Statistically valid plate counts are only obtained from bacterial dilutions that yield between 30 and 300 colonies. 

Picture of Quebec Colony Counter

E-portfolio week 11

Experiment 16 and 17
Experiment 16: Physical Factores: Atmospheric Oxygen Requirements
In this experiment, I have learnt different oxygen requirements of different microorganisms. Basically, microorganisms are divided into five groups based on their oxygen requirements. There are aerobes which require oxygen, microaerophiles which require limites amounts of atmospheric oxygen for growth, obligate anaerobes which requires absent of oxygen, aerotolerant anaerobes whivh are fermentative organisms and facultative anaerobes which can grow in the presence or absence of oxygen. To carry out this experiment, the procedures are as followed.The procedures of the experiment are as followed. The sterile brain heart infusion agar is first liquefied by boiling in a waterbath at 100°C. Molten agar is cooled to 45°C. The temperature is checked with a thermometer inserted into the waterbath. To determining oxygen requirements, the following steps are taken.Using aseptic technique, each experimental organism is inoculated by introducing two drops of the culture that is Staphylococcus aureus, Corynebacterium xerosis and Enterococcus faecalis, Saccharomyces cerevisiae, Aspergillus niger and Clostridium sporogenes from a sterile Pasteur pipette into the appropriately labeled tubes of molten agar. The freshly inoculated molten infusion agar is vigorously rotate between the palms of the hands to distribute the organisms. Inoculated test tubes is placed in an upright position in the iced waterbath to solidify the medium rapidly. The Staphylococcus aureus, Corynebacterium xerosis, Enterococcus faecalis and  Clostridium sporogenes cultures is incubated for 24 to 48 hours at 37°C and the Aspergillus niger and Saccharomyces cerevisiae cultures is incubated for 48 to 72 hours at 25°C. After incubation, each of the experimental cultures is observed for the distribution of growth in each tube. Observation is recorded and the oxygen requirements for each of the experimental species is determined in the chart provided in the Lab Report.

Picture of C. sporogenes which is aerotolerant

Experiment 17:Techinques for the Cultivation of Anaerobic Microorganisms
From this experiment, I learnt that some microorganisms can only survive under anaerobic microorganisms. I learnt how to determine anaerobes and aerobes through this experiment. The procedures of the experiment are as followed.The procedures of the experiments are as followed. Firstly, the experiment is carried out by using the fluid Thioglycollate Medium. For the performance of this procedure, the fluid thioglycollate medium must be fresh. Freshness is indicated by the absence of a pink color in the upper one- third of the medium. If this coloration is present, the screw caps is loosen and the tubes is placed in a boiling water bath for 10 minutes to drive off the dissolved O₂ from the medium. The tubes is cooled to 45°C before inoculation. The appropriately labeled tubes organisms that is the Bacillus cereus, Escherichia coli, and Micrococcus luteus and Clostridium sporogenes is aseptically inoculated by means of loop inoculations to the depths of the media. The cultures are incubated for 24 to 48 hours at 37°C.

 Secondly, the experiment is carried out using GasPak Anaerobic Technique. The GasPak system is a contemporary method for the exclusion of oxygen from a sealed jar used for incubation of anaerobic cultures in a nonreducing medium. This system uses a GasPak generator that consists of a foil package that generated hydrogen and carbon dioxide upon the addition of water, A palladium catalyst in the lid of the jar combines the evolved hydrogen with residual oxygen to form water, thereby creating a carbon dioxide environment within the jar that is conducive for anaerobic growth. The establishment of anaerobic conditions is verified by the colour change of a methylene blue indicator strip in the jar. This blue indicator becomes colourless in the absence of oxygen.

The procedures of GasPak Anaerobic Technique are as followed. By using a glassware marking pencil, the bottom of each nutrient agar plate is divided into two sections. Each section on two plates is labelled with the name of the organism to be inoculated that is Bacillus cereus, Escherichia coli, and Micrococcus luteus and Clostridium sporogenes. Step 2 is repeated to prepare a duplicate set of cultures. Using aseptic technique, a straight line streak of each test organism is made in its respectively labelled section on both sets of plates. The corner of the hydrogen and carbon dioxide gas generator is torn off and this is inserted inside the GasPak jar. One set of plate cultures is placed in an inverted position inside the GasPak chamber. The anaerobic indicator strip is exposed and is placed inside the anaerobic jar so that the wick is visible from the outside. With a pipette, the required 10ml of water is added to the gas generator and the chamber is quickly sealed with its lid. The sealed jar is placed in an incubator at 37°C for 24 to 48 hours. After several hours of incubation, the indicator strip is observed for a colour change to colourless, which is indicative of anaerobic conditions. The duplicate set of plates is incubated in an inverted position for 24 to 48 hours at 37°C under aerobic conditions. After incubation, the Fluid Thioglycollate cultures, GasPak system, and aerobically incubated plate cultures are observed for the presence of growth. Results are recorded in the chart provided in the Lab Report. Based on observation, the oxygen requirement classification of each test organism is recorded as anaerobes, facultative anaerobes, or aerobes.

Picture of microorganisms in agar plate to study the oxygen requirements.