LAB 3 : Preparation and Sterilization of Culture Media

Introduction :

            Culture media are available commercially as powders; the require only addition of water. Nutrient medium is a general preparation for culturing microorganisms which are not nutritionally fastidious.The broth contains:

1.50 g/L "Lab-lemco" powder (a beef extract)
1.50 g/L yeast extract
5.00 g/L peptone （a nitrogen source)
5.00 g/L sodium chloride
15.00 g/L agar powder

The agar has the same composition, except that it contains 15g/L agar. The final pH of both media is 7.4.

     Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121^oC for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurized steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented.

     Steam is continually forced into the chamber until the pressure reaches 103kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121^oC. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121^oC throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilized objects can then be removed.

Objective :

To prepare sterile nutrient agar for culturing microorganisms.

Materials and reagents :

Commercial nutrient agar

Balance

Distilled water

Scott bottles

Measuring cylinder

Beaker

Glass rod

Procedures :

1. The amount of broth ( with agar ) is weighed appropriately in the Scott bottles and is  dissolved with distilled water. The mixtureis then mixed well.
2. The bottles is loosely recap and set aside for sterilization.
3. All media is sterilized at 121^oC for 15 minutes.
4.  After autoclaving, the media is removed. The broth preparation is allowed to cool then the cap of each bottle is tighten.

Discussions :

1. Precautions 

a)     The pan and the balance must be cleaned before weight the culture medium powders. This is to ensure that more accurate amount of culture medium powders is weighed.

b)     The door of the balance machine should also be closed before weighing the substances and pressed the ‘tare’ button after the pan or beaker is put onto the balance to avoid zero error.

c)     Before use the apparatus, rinsed all the apparatus with distilled water to avoid contamination.

d)     The Scott bottles` cap have been heated in autoclave should be recapped loosely before putting into the autoclave machine. This is to prevent the Scott bottles from breaking during autoclaving process.

e)     Use the rod to stir before pour the media into the Scott bottles to ensure that all powders from cultural medium were dissolved in the distilled water.

2. Working of Autoclave 

     Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained. As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure. The temperature rises to 121.5 ^oC  and the superheated water molecules rapidly conduct heat into microorganisms. 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.

3. Effectiveness of Autoclave or Optimum Conditions

    Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121 ^oC) will kill all organisms and their endospores in about 15 minutes.

Conclusion :
The process that we go through when preparing culture media are important before autoclaving. We are able to prepare culture medium with correct quantities of ingredients. Autoclaving is the effective way use in sterilization.



References :  
http://microbiologyon-line.blogspot.com/2009/08/autoclaving-real-sterilization.html
http://en.wikipedia.org/wiki/Autoclave
http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html

LAB 1 : Principles and Use of Microscope ( Part II )

B. Examination of cells

　　Introduction：

        Bacteria are not generally studied with the low-power or high power-power dry objectives because of their extreme minutes. Instead they are stained and observed with the oil immersion objective.

 　　The wet mount method enables you to study the sizes and shapes of the living microorganisms (drying or straining microorganism distort them). It also enables you to determine if cells are motile. The wet mount method is quick and easy, and does not require special equipment.　

　　Objective：

•          To provide an experience in the use of microscope．
•          To illustrate the diversity of cells and microorganisms．

　　　

　　　Materials and reagents：

• 　Culture
• 　Immersion oil
• 　Lens tissue
• 　Inoculating loop
• 　Bunsen burner
• 　Slide and cover slip

            Procedure: 

1. Bunsen burner is lighted up.
2. One hand is used to sterile the inoculating loop. Then leave it to cool down.
3. Other hand was used to hold the culture. With the same hand replace the culture with slide.
4. Inoculating loop is used to transfer culture into the slide.
5. The inoculating loop is put down and places the edge of cover slip onto the slide and lowered it gently to cover the drop of culture.
6. The slide is placed to the microscope stage and observed by using 4x, 10x, and 40x objective focus on the culture.
7. Immersion oil is applied onto the slide to be observed under 100x magnification.
8. Culture is observed was drawn.
9. This procedure is repeated with other culture.

                 

                 Result : 

             

                 The slide

       Observation :

      Lactobacillus sp. 

  
   From Observer :

Video :

Escherichia coli

From Observer :

Video :

        Discussion :

        Bacteria usually studied with the low-power dry objective. But in order to study their movement, size and shape, the wet mount must be used. This is because technique in preparing wet mount does not kill the bacteria. Simple technique of aseptically is used to transferred the bacteria from medium bottle to glass slide. Then, immersion oil is used at 100x objective lens unless the cell is very small and moving slowly.

The bacteria:

1. Lactobacillus acidophilus

The scientific classification

·         Kingdom : Bacteria

·         Division : Firmicutes

·         Class : Bacilli

·         Order : Lactobacillales

·         Family : Lactobasillaceae

·         Genus : Lactobacillus

·         Species : L.acidophilus

The morphology

·         Shape : rod shape

·         Color : grey

·         Size : 0.6-0.9 μm in width and 1.5-6.0 μm in length

2. Escherichia coli

The scientific classification

·         Kingdom : Bacteria

·         Division : Eubacteria

·         Class : Proteobacteria

·         Order : Enterobacteriales

·         Family : Enterobacteriaceaa

·         Genus : Escherichia

·         Species : E. coli

The morphology

·         Shape : rod shape

·         Color : red

·         Size : approximately 0.5 μm in width by 2 μm in length

References

1.              http://www.microbehunter.com/2010/08/13/making-a-wet-mount-microscope-slide/
2.              http://www.trueart.info/wet_mounting.htm
3.               http://en.wikipedia.org/wiki/Lactobacillus
4.               http://en.wikipedia.org/wiki/Lactobacillus_acidophilus
5.  .            http://en.wikipedia.org/wiki/Escherichia_coli

LAB 1 : Principles and Use of Microscope ( Part I )

A. Setting up and using the microscope

      　Introduction:

      　In order to be seen, microorganisms need to be magnified. Despite advances in other area of microscopy (for example, the electron microscope), the light microscope is still the instrument most frequently used for viewing microorganisms.

            Objective:

            Learn to use a simple bright-field microscope correctly.

            Materials:

            Microscope slide, cover-slip, specimen slide

           Procedures :

1.  The microscope is placed at a comfortable position to make sure the observation is more easy.
2. The main switch of the microscope is then turn on.
3. The light intensity is then adjusted using the brightness control.
4. The revolving nosepiece is rotated to bring the 4X objective lens into the light path.
5. A line is marked on a clean slide with a marker pen. The slide is then placed on the stage of the microscope and secure by spring clip. The slide is moved to the light path using the coaxial stage control knobs.
6. Both of the eyepieces is adjusted until a single circle light is been seen.
7. The tube length adjustment ( diopter) ring on the right eyepiece is adjusted to match interpupillary distance .
8. The marker-pen mark is focused by adjusting the coarse and fine adjustment knobs and observed through right eye.
9. The left eyepiece is then focused using the tube lens adjustment          (diopter) ring and observed through left eye.

        

        Using microscope objective viewing:

1. Marker pen slide is replaced with specimen slide.
2. By watching at the side of microscope, nosepiece was rotated to 10x objective lens.
3. The specimen is focused using fine adjustment knob and by moving the stage.
4. Condenser and light intensity were adjusted to obtain better image.
5. The image of the specimen was capture.
6. Step 2 to step 5 is repeated by using 40x and 100x objective lens.

      Results:

      

      The slide

     Observation :

     Penicilium sp under 4x magnification

    Penicilium sp under 10x magnification

    Penicilium sp under 40x magnification

        Discussion :

      By using the magnification of 4x, 10x, and 40x we are able to see a very clear image of the penicillium sp. The specimen is observed from the lowest 4x magnification to the highest magnification which is 40x. We can see clearly the morphology of penicillium sp. The penicillium sp was no need to observe under the oil immersion(100x) objective lens because it is type of fungi.

Scientific classification of penicillium sp. :

·         Kingdom : fungi

·         Class : eurotiomycetes

·         Order : trichocomaceae

·         Genus : Penicillium 

     

     Morphology

     o  Colour:  Red

     o  Shape:  brush-like structure        

     o  Surface: rough

       References :

           ~http://en.wikipedia.org/wiki/Penicillium#Characteristics

         ~http://www.caltexmoldservices.com/section/mold_library/penicillium/

Behind the Scenes

We really have a lot of FUN with each of our members when doing our First lab~~

We have learn a lot of new things,

especially the uses of the MICROSCOPE~~

All our eyes feel so tired when try to observe the microorganism under the microscope

The MOST FuN time comes when we faced the Neubauer Chamber

This Small thing make us CRAZY as we need to observe the Grid on it!!!

we waste most of our Time to observe it~

The Funniest part is that we make the same MISTAKE Twice!!!

OMG!!!

We use wrong COVERSLIP @@

Can`t imagine that we waste so much time to observe nothing~

our eyes already wanna POP OUT~

We do not give up,

we change the correct coverslip and try the find the Grip again...

Trying...

Trying...

Everyone is trying...

Finally,

We saw an excited face from Hafis,

His eyes became bigger and bigger...

And

His sunshine SMILE~~

YEAH~~!!!!

We get it!!

All of us feel so HAPPY~~~

Luckily we do not give up,hehe~~~

We Are so Professional,

We have the Spirit of Scientist!

Happy captured for our First LaB~~

LAB 2: Measurement and Counting of Cells Using Microscope

2.1 Ocular Micrometer

            Introduction :

            Ocular micrometer is use in order to measure and compare the size of prokaryotic and eukaryotic microorganisms. Microorganisms are measured with an ocular micrometer which is inserted into the one of the microscope eyepieces. The micrometer, which serves as a scale or rule, is a flat circle of glass upon which are etched equally spaced divisions.This is not calibrated, and may be used at several magnifications. When placed in the   eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed maybe calibrated using a stage micrometer on which parallel lines exactly 10μm apart etched. By determining how many units of the ocular micrometer superimpose a known distance on the stage micrometer, you can calculate the exact distance each ocular division measures on the microscopic field. When you change objectives you must recalibrate the system. After calibration of the ocular micrometer, the    stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may then be determined.

            Objective :

            To measure and count cells using a microscope.

            Materials :

            Light microscope

            Ocular micrometer

            Stage scale

            E.coli slide

                Methods :

1. One of the eyepiece on the light microscope is changed with the eyepiece that contains the ocular    micrometer.
2. The stage scale is then put on the stage of the microscope.
3. Start from the lowest power objective (4X) , focus the microscope until the image on the stage scale is superimposed with the eyepiece scale.
4. The number of divisions on the ocular micrometer is determined and calculated.
5. The procedure is repeated when using high power magnification.

Results :

4.0 ocular micrometer = 0.01 mm stage micrometer ( 40 x magnification )

9.6 ocular micrometer = 0.01 mm stage micrometer ( 100 x magnification )

4.1 ocular micrometer = 0.001 mm stage micrometer ( 400 x magnification )

Lactobacillus

Discussion :

1. Ocular micrometer has no units on it.
2. We have to make sure 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.
3. The primary challenge is to measure the size of one Lactobacillus cell from our sample.
4. For our experiment, we are able to calibrate a scale at 400x magnification which is already enough to be used to convert into other magnifications.
5. The actual size of the Lactobacillus sample through our observation is 1.708x10^-4mm.

Reference :

1. http://en.wikipedia.org/wiki/Ocular_micrometer
2. http://wiki.answers.com/Q/Why_is_it_necesary_to_calibrate_the_ocular_micrometer_with_each_objective
3. http://www.ruf.rice.edu/~bioslabs/methods/microscopy/measuring.html

2.2 Neubauer Chamber

Introduction :

                Neubauer chambers are more convenient for counting microbes. The neubauer is a       heavy glass slide with two counting areas separated by a H-shaped trough. A special            coverslip is placed over the counting areas and sits a precise distance above them.

Objective :
To measure and count cells using a microscope.

Materials :
Light microscope
Neubauer chamber       

Coverslip

Bunsen burner
Sterile Pasteur pipettes

Lactose bacillus culture

Methods :

1. Lactose bacillus culture is added to the space between the coverslip and the counting chamber with the use of sterile Pasteur pipettes.
2. The cells is allowed to settle about one minutes.
3. The number of cells on the counting chamber is then observed and counted under the microscope.
4. The procedure is repeated when using high power magnification.
5. After finish the experiment, the Neubauer and coverslip is cleaned with 70% alcohol.

Results :

Figure 1 . Neubauer Chamber 

Figure 2

Figure 2 and 3 . Neubauer Chamber 's Grids

           Discussions :

           The neubauer  chamber`s grip common was 24 x 16. Mostly use to observe the blood     
           cell and now also being use to calculate concentration or density of others cell.
 

1. The depth of the chamber was 0.1 mm, Therefore the volume = 0.04mm² x 0.1mm     = 0.004mm³ = 4x10^-3mm³ = 4x10^-6cm³
2. The most important things is use the correct cover (neubauer counting chamber cover glass). If not you will not able to see the grip.
3. If lose count when counting the cell, has to recount one more time.
4. Count the cell immediately after loading, because some cell may aggregate and this will affect the result.

References :

1. http://shop.gabsystem.com/data/descargas/Camara%20thoma%20neubauer_Eng.pdf
2. http://people.oregonstate.edu/~weisv/Protocols/Symbiodinium/Cell%20Counts.pdf
3. http://wiki.answers.com/Q/Why_is_70_percent_alcohol_a_better_disinfectant_than_100_percent_alcohol