Saturday 24 October 2015

LAB 3 :PREPARATION AND STERILIZATION OF CULTURE MEDIA


INTRODUCTION

Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have potential to adapt to the habitats most suitable for their needs, but then it is hard for microbes to do so  in the laboratory. This is when a growth media or also known as culture media plays an important part.  This is basically an aqueous solution to which all the necessary nutrients have been added, depending on the type and combination of nutrients, different categories of media can be made. A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells. Different types of cells prefers different type of media, depending on their needs. There are actually two major types of culture media, one of it are those used for cell culture, which use specific cell types derived from plants or animals. The second one is microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates.


The composition of self-made agar broth is listed below :


1.5 g/L “Lab-lemco” powder (a beef extract)
1.5 g/L Yeast extract
5.0 g/L Peptone (a nitrogen source)
5.0 g/L Sodium chloride
15.0 g/L Agar powder
For your information, the self-made agar broth actually contains the same composition with the manually-made nutrient medium, except that it contains 15 g/L agar. We must also ensure that the final pH value of both medias is 7.4 .


Speaking of autoclaving, autoclaves are more or less like pressure cookers very similar to the ones that you see in the stores. As we know, food cook a lot faster in a pressure cooker than they do in a regular pot or in the oven. This is due to the intense heat and pressure that is applied to the food. The same mechanism works against living microorganisms in an autoclave. Once an autoclave is started, steam is pushed into the chamber that contains the items that are being sterilized. As the steam goes in, the pressure and temperature within the chamber is increased. Most autoclaves are set to increase steam pressure until a temperature of at least 121 degrees Celsius is reached. This temperature and pressure will remain at this level for at least 15 minutes. This is a high enough temperature for a long enough period of time to kill any and all microorganisms and their spores. The steam and pressure are released and brought down to normal room temperature and pressure after the 15 or more minutes of running.

     Image 1 : Example of culture media in agar plates

OBJECTIVE
To prepare sterile nutrient agar for culturing microorganisms .


MATERIALS AND REAGENTS
Commercial Nutrient Agar
Brain Heart Infusion Broth ( BHI )
Trypticase Soy Broth ( TSAYE )
Peptone powder
Beef extract powder
Sodium chloride
Yeast extract
Electronic Weighing Balance
Distilled water
Scott bottles
Measuring cylinder
Glass rod
Beakers
PROCEDURE

A . Commercial nutrient agar
1 . 11.2 g of the commercial nutrient agar is weighted using an electronic
    weighing balance and placed into a beaker.
2 . 400 ml of distilled water is measured using a measuring cylinder and poured into
    the beaker containing the nutrient agar. The solution is then stirred by using a
     glass rod until it mixes well.
3 . After the solution is mixed well, the solution is poured into the Scott bottle that
    had been sterilized.
4 . The bottle is the loosely recapped and is set aside and ready to undergo
    sterilization in an autoclave machine.
5 . The media is sterilized at 121 degree Celsius for 15 minutes.
6 . The media is removed after 15 minutes of autoclaving. The media is allowed to
     cool down and the cap of the bottle is tighten.


B . Self-made nutrient agar
1 . 0.60 g of beef extract, 0.6 g of yeast extract, 2.0 g of peptone, 2.0 g of sodium
    chloride and 6.0 g of agar powder are weighed using an electronic weighing
    balance and placed into a beaker.
2 . 400 ml of distilled water is measured using a measuring cylinder and poured into
    the beaker containing the nutrient agar. The solution is then stirred by using a
    glass rod until it mixes well.
3 . After the solution is mixed well, the solution is poured into the Scott bottle that
    had been sterilized.
4 . The bottle is the loosely recapped and is set aside and ready to undergo
    sterilization in an autoclave machine.
5 . The media is sterilized at 121 degree Celsius for 15 minutes.
6 . The media is removed after 15 minutes of autoclaving. The media is allowed to
     cool down and the cap of the bottle is tighten.


C . Brain Heart Infusion agar (BHI)
1 . 5.20 g of BHI agar in powder form is weighed using an electronic weighing
    balance and placed into a beaker.
2 . 100 ml of distilled water is measured using a measuring cylinder and poured into
    the beaker containing the nutrient agar. The solution is then stirred by using a
    glass rod until it mixes well.
3 . After the solution is mixed well, the solution is poured into the Scott bottle that
    had been sterilized.
4 . The bottle is the loosely recapped and is set aside and ready to undergo
    sterilization in an autoclave machine.
5 . The media is sterilized at 121 degree Celsius for 15 minutes.
6 . The media is removed after 15 minutes of autoclaving. The media is allowed to
     cool down and the cap of the bottle is tighten.

D . Trypticase Soy Agar ( TSAYE )
1 . 4.00 g of TSAYE agar in powder form is weighed using an electronic weighing
    balance and placed into a beaker.
2 . 100 ml of distilled water is measured using a measuring cylinder and poured into
    the beaker containing the nutrient agar. The solution is then stirred by using a
    glass rod until it mixes well.
3 . After the solution is mixed well, the solution is poured into the Scott bottle that
    had been sterilized.
4 . The bottle is the loosely recapped and is set aside and ready to undergo
    sterilization in an autoclave machine.
5 . The media is sterilized at 121 degree Celsius for 15 minutes.
6 . The media is removed after 15 minutes of autoclaving. The media is allowed to
     cool down and the cap of the bottle is tighten.


RESULTS
4 different culture media was prepared which are 400 ml of commercial nutrient agar, 400 ml of self-made nutrient agar, 100 ml of Brain Heart Infusion (BHI) agar and 100 ml of Trypticase Soy Agar ( TSAYE ). The composition of the materials needed are stated in the procedure. They are weighed approximately and dissolved with distilled water. Stirring of the solution takes place until the solution is dissolved and mixed. After mixing, it is only poured into sterilized Scott bottles and ready to place into an autoclave machine for 15 minutes at the temperature of 121 degree Celsius.

Image 2 : Weighting the appropriate amount using an electronic weighting balance

Figure 3 : Done weighing the 6.00 g agar powder for self-made nutrient agar

Figure 4 : The preparation for self-made nutrient agar
Figure 5 : Appropriate amount of distilled water obtained using a measuring cylinder

Figure 6 : Transfer of the solution after mixing with distilled water into Scott bottles 


Figure 7 : Four different cultured media prepared

Figure 8 : Cultured media ready to undergo autoclaving

Figure 9 : Cultured media in an autoclave
DISCUSSION

1 . There are actually a few precautions that we need to take note throughout the
     experiment, which is :
  • The pan of the electronic weighing balance is cleaned with a small brush first to prevent any small leftover particles that might affect the weight reading.
  • The “tare” button is pressed every time after the empty beaker is put on the balance to obtain accurate measurements and to prevent zero errors.
  • When using a measuring cylinder to obtain distilled water, make sure that the position of the eye is at the same level as the bottom of the meniscus ( surface of water that is curved downwards ) to prevent parallax errors.
  • All of the apparatus used are cleaned and rinsed using distilled water before using.
  • The media is stirred well using a glass rod to ensure balance mixing and to maintain the concentration of the media.
  • Make sure that the caps of the Scott bottles are only slightly tightened to prevent the Scott bottles from breaking during autoclaving.


2 . Before the Scott bottles with different medium are placed into the autoclaving machine for sterilization, there are some steps that need to be followed as below :
  • The drain screen at the bottom of the chamber is checked before using the autoclave.
  • Any debris noticed is cleaned up for efficient heat transfer as steam must flush out of the autoclave chamber. If the drain screen is blocked with debris, a layer of air may form at the bottom of the autoclave and prevent proper operation.
  • The water level is ensured to be higher than the bottles in the autoclave.  
  • The cover of the autoclave chamber and exhaust valve is tightened.
  • The temperature is checked so it is always maintained at 121°C and the pressure is ensured to reach 103 kPa above the atmospheric pressure, with steam is continuously forced into the chamber.
  • The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur.
  • Then, the exhaust valve is opened to ensure the pressure drops to nearly 0 kPa before removing the basket with Scott bottles from the autoclave chamber.
CONCLUSION

As a conclusion, we are able to learn the correct steps and methods to prepare different media for culturing microorganisms. Precautions must also remembered when carrying out the steps and procedures.  Preparation and sterilization of culture media is important to prevent contamination of the unwanted microorganisms inside the media. We also obtained the information that autoclaving is actually a fast and efficient sterilization process.   

Reference

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Monday 12 October 2015

LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE


2.1 Ocular Micrometer
INTRODUCTION
An ocular micrometer is a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects. In other words , it serves as a scale or ruler to measure the size of magnified objects. The physical length of the marks on the scale depends on the degree of magnification. The ruler on a typical ocular micrometer has between 50 to 100 individual marks, is 2 mm long and has a distance of 0.01 mm between marks. Technicians can easily make calculations of object size after measuring an object against the dimensions of an ocular micrometer, which are calibrated using a stage micrometer, a microscope slide with its own surface scale visible when viewed through the microscope . The function of the stage micrometer is to achieve calibration of the microscope by checking  the divisions and measurements on the eyepiece reticle. The distance between the lines of an ocular micrometer is an arbitrary measurement that only has meaning if the ocular micrometer is calibrated for the objective being used. In the world of science, an ocular micrometer is usually used to measure the sizes of prokaryotes and eukaryotes. It is also used to categorized newly found microorganisms and distinguish them based on their sizes .


Figure 1 : Micrometer eyepiece

Figure 2 : An Ocular micrometer


OBJECTIVE
1 . To learn the correct steps and procedures of using an ocular micrometer.
2 . To measure and count cells using a microscope
MATERIALS AND REAGENTS
Microscope fitted with an ocular micrometer , slide micrometer and stained preparation.

PROCEDURE
( Refer to manual )


RESULTS

The ratio of magnification is calculated by using the formula below,

   One ocular division = No. of division on stage micrometer
                                      No. of division on ocular micrometer
When using 10 x objective lens , 1 mm on stage represent 9.6 ocular divisions
When using 40 x objective lens , 0.1 mm on stage represent 8.0 ocular divisions
For 40 x objective lens ,
The length of cells : 8.0 ocular unit = 0.1 mm
                               0.2 ocular unit = 2.50 µm
The width of cells : 8.0 ocular unit = 0.1 mm
                              0.1 ocular unit =1.25 µm


Figure 3 : 40 x objective lens , 400 maginification
Figure 4

DISCUSSION
1 . We have to ensure that the first line of the ocular micrometer is in line with the
    first line of the stage micrometer to avoid errors when reading the measurements,
     this is what be call calibration .
2 .  In this experiment, parallax errors should be prevented when calibrating the
     ocular micrometer with the stage micrometer to get an accurate calibration.
3 . An ocular micrometer does not have any units, therefore it is a must for it to be    
    calibrate with the stage micrometer to get the correct unit and scale.
REFERENCE
the 10th of October 2015


2.2 Neubauer Chamber

Neubauer chamber also known as a hemocytometer is a device used to count cells and was originally designed for counting red blood cells. Touching a little on the history of the hemocytomer, it was actually invented by Louis-Charles Malassez. The Neubauer chamber is a thick crystal slide with the size of a glass slide of  30 x 70 mm and 4 mm thickness . A neubauer chamber consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The device is carefully crafted so that the area bounded by the lines and depth is known. It is therefore possible to count the number of cellsor particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall. In a simple counting chamber, the central area is where cell counts are performed. The chamber has three parts. The central part, where the counting grid has been set on the glass. Double chambers are most common than simple chamber. In this case, the chamber has two counting areas than can be loaded  mm in size. The grid has 9 square subdivisions of width 1 mm.  Cover slips for counting chambers are specially made thicker than those for conventional microscopy, since they must be heavy enough to overcome surface tension of a drop of liquid. The cover slip is placed over the counting surface prior to putting on the cell suspension.

Figure 5 : A nerbauer chamber

OBJECTIVE
1 . To practice how to use a neubauer chamber precisely to count the cell .

MATERIALS AND REAGENTS
Cell line culture, neubauer chamber and cover slip, Sterile Pastuer pipettes .

PROCEDURE

( Refer to manual )

RESULTS
Total number of cells in 9 random small squares  = 5+6+5+5+6+5+6+4+4
                                                  = 46
The average number of cells per square = 46 / 9
                            = 5.11 cells
Volume of the square = 0.25 mm x 0.25 mm x 0.1 mm
              = 6.25 x 10-3 mm 3
              =  6.25 x 10-6 cm
              = 6.25 x 10-6  mL
This shows that there are 5.11 cells in 6.25 x 10-6  mL , therefore the concentration of cells = 5.11 cells / 6.25 x 10 -6 mL  
= 8.176 x 10 5 cells / mL

Figure 6 : Image of cell line on hemacytometer


DISCUSSION
1 . A few drops of cell line culture is transfered to the surface of the hemocytometer
    by using aseptic techniques practiced in the previous lab. The specially made   
    cover slip is a must to be placed on the hemocytometer to give a precise volume
    in the space delimited by the grid and the cover slip.
2 . The cover slip must be placed on the surface of  hemocytometer carefully to avoid
    the air bubbles from forming .This is because the air bubbles actually block our
    vision when examining the cell in the counting chamber. Cover slip for counting
    chambers is specially made and thicker than those for conventional microscopy.    
    Since they must be heavy enough to overcome the surface tension of a drop of
    liquid.
3 . 9 small squares are selected randomly without being picky from a total of 25
    square to get an accurate result . If any of the cells are located between two
    boxes , it should be counted only once but not twice . It is also advisable not to     
    count cells which are further expose outside the square .
4 . Then at last, the average number of cells is obtained and the concentration of
     cells can be calculated .


CONCLUSION
With the help of an ocular micrometer, we are able to measure magnified specimens or microorganisms under the microscope. On the other hand, with the help of a Neubauer chamber or hemocytometer, we are able to calculate the cell concentration in a culture and get a rough number of cells in the culture.

REFERENCE
2 . Obtained from https://en.wikipedia.org/wiki/Hemocytometer on the 10th of October 2015  

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