LAB 3 Preparation and Sterillization of Culture Media

Introduction

There is a great deal of nutritional diversity among microorganisms. Therefore, microbial growth is greatly affected by the nutrients that are available in their environment. As such a culture media, which purpose is to provide nutrient for the growth of microorganisms or cells must be prepared and sterilize thoroughly.

Culture media that is often used in a laboratory setting can be classified into two (2), a complex media or a defined media. Complex media are composed of extracts from biological sources which components are unknown while a defined media is a media with known constituents. For this lab, preparation of sterile nutrient agar for culturing microorganisms will be the main objectives.

Although there is nutrient agar which is available commercially, it is also vital for a student in microbiology to know what constitute a culture media. Thus, several type of culture media will be prepared which includes BHI agar, TSAYE agar, commercial nutrient agar and a self-made nutrient agar. The self-made nutrient agar will contains:

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 

It should also be known that the nutrient agar is initially a liquid media or a nutrient broth which is mixed with agar and poured via sterile media dispenser into Petri dishes to solidify.

Next, a culture media can be sterilized using a process known as autoclaving. It is proceeds with an autoclave which is basically a pressure chamber that is sealed off against surrounding air and functions similarly like a large pressure cooker. The exact conditions for autoclaving includes temperature of 121 °C under the pressure of 15 psi or 103 kPa for 15 minutes.

Objective

To prepare sterile nutrient agar for culturing microorganisms

Materials and reagents

Commercial nutrient agar
Balance
Distilled water
Scott bottles

Procedure

(refer to lab manual)

Results

4 culture media has been prepared. There are 400 ml of nutrient agar, 400 ml of self-made nutrient agar, 100 ml of BHI agar and 100 ml of TSAYE agar.

For the self-made nutrient agar, the recipe is as stated below:

‘Lab-lemco powder’ (a beef extract)  0.6 g/ml

Yeast extract                                       0.6 g/ml

Peptone                                               2.0 g/ml

Sodium chloride                                 2.0 g/ml

Agar powder                                       6.0g /ml   
  

Auto-claving

Discussion

- The surface of the balance must be cleaned before weighing the culture media powder to get more accurate reading.

- All of the doors of the electronic balance are closed before weighing.

- The draft shield is closed completely and zero using the "Tare" key on balance display. This will clear any previous tare or cancel any zero-drift.

- All the apparatus are cleaned with distilled water before used to avoid contamination.

-The Scott bottles are labeled with the name of culture media (nutrient agar, self-made nutrient agar, BHI agar and TSAYE agar) so that it easy for us to differentiate them.

- All of the media are stirred well with a spatula to ensure that the powders from cultural medium mixed well with the distilled water.

-The caps of the Scott bottles are recapped loosely to prevent the Scott bottles from breaking during the autoclaving.

-All of the Scott bottles which contain the different medium are placed into a special basket then the whole basket is put into the autoclave chamber for sterilization.

-All the media are sterilized at 121 °C and 103 kPa (15psi) for 15 minutes. Under these conditions, all organisms and their endospores will be killed.

-After autoclaving, the exhaust of autoclaving machine must be opened to lower the pressure until the pressure is same as atmospheric pressure.

Conclusion

The preparation of nutrient agar for the culturing microorganism is done by using autoclaving technique in order the material to be sterilized. Higher temperature ensure more rapid killing. It is very important to ensure that all the trapped air is removed.
Proper autoclaved treatment will inactive all fungi bacteria, viruses and also bacterial spores which can be quite resistant, if not necessarily eliminate all prions.

Reference

• http://microbiologyon-line.blogspot.my/2009/08/autoclaving-real-sterilization.html
• http://us.mt.com/us/en/home/supportive_content/tips_and_tricks/CSM_LAB_Ciesniewski_Weighing_Skills.html
• http://www.ibg102.blogspot.my/2014/12/lab-3-preparation-and-sterilization-of.html?=1

Lab 2

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. The physical length of the marks on the scale depends on the degree of magnification. 

Formula: Micrometer pitch length(under microscope)= 

Actual micrometer pitch length/Objective lens magnification

Objectives

To measure and count the cells using a microscope 

Materials and reagents

Microscope fitted with an ocular microscope

Slide micrometer

Stained preparation of yeast and bacteria

Procedure

(refer to lab manual)

Results

Ocular Micrometer

Stage Micrometer

The scale of Ocular Micrometer and Stage Micrometer

Calculation:

All division of stage micrometer = 1mm =1000 micrometer (µm).

10x objective lens : 9.5 ocular units = 1mm.

40x objective lens : 3.9 ocular units = 0.1 mm.

The length of the yeast.

10x objectives lens : 0.1 ocular units = 0.0105 mm=  10.5 µm.

The width of the yeast.

10x objectives lens : 0.1 ocular units = 0.0105 mm=  10.5 µm.

Discussion

The dimensions of cells may be determined using an ocular micrometer. However, several steps must be completed first before microorganisms can be measured. This is because, the ocular micrometer is not calibrated with the microscopic field and with the help of stage micrometer this can be corrected. When calibrating, the scale of ocular micrometer need to be adjusted and superimposed on a parallel line with the stage micrometer. A known distance of ocular unit on the stage micrometer will be used for calculations.

1. After calibrating and using 10x objective lens it is determined that 1mm of stage scale is equal to 9.5 ocular units.
2.  Next, using the 40x objective lens and after recalibrate the system it is determined that 3.9 ocular units is equal to 0.1 mm based on the stage scale.
3. Therefore, the length of yeast is 0.0105 mm or 10.5 µm as it is measured to be 0.1 ocular unit when using the 10x objectives lens.
4.  The width of the yeast is similar to its length as it is almost round in shape and also measured to be 0.1 ocular units.

Conclusion

From the experiment, the length and width of the yeast cells is 10.5 micrometer. By using ocular micrometer and stage micrometer, the size of the microorganism like yeast can be measured.

2.2 Neubauer Chamber 

Introduction

Neubauer Chamber which is called as hemocytometer is a device used to count cells. It was originally designed for the counting of blood cells.

The hemocytometer was invented by Loius-Charles Malassez and 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 which produce 9 major large squares. The device is carefully crafted so that the area bounded by the lines is known and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid and thereby calculate the concentration of cells in the fluid overall.

Materials and reagents

Serial dilutions of bacteria cultures

Neubauer and coverslip

70% ethanol

Sterile Pasteur pipettes

Procedure

(refer lab manual)

Results

Yeast was observed using hemocytometer

Total cells in 8 small boxes =  116 cells.

Average cells for 1 small boxes : 116 cells  /8 = 14.5 cells.

Volume of one small box = 0.25 mm x 0.25 mm x 0.1 mm = 6.25 x 10^-3 mm³

                                                                                      =6.25 x 10^-3 x 0.001 cm ³

                                                                                       = 6.25 x 10^-6 cm³

                                                                                        = 6.25 x 10^-6 mL.

Dilution of yeast culture = 10 times.

Cell concentration = 14.5 cells / (6.25 x 10^-6mL).

                                  = 2320000 cells =2.32 x 10⁶ cells per mL
After dilution, cell concentration = 2.32 x 10⁷ cells per mL

Discussion

Diluted yeast culture of 10 times was used for this experiment as the culture is very concentrated and a drop of it is put between the coverslip and counting chamber of hemocytometer. This step are conducted using aseptic technique to ensure sterility

1. The chamber used contains many grids but only the middle large box is use for calculation.
2. The middle large box has a size of 1mm x 1mm with a depth of 0.1 mm and contains 16 smaller boxes.
3. This means that the volume of 1 small box is 0.25mm x 0.25mm x 0.1 mm which is equal to 6.25 x 10^-3 mm³ or 6.25 x 10^-6 mL when converted.
4.  8 small boxes are chosen out of 16 and the average cells in 1 small box are calculated.

 By using the formula :   

Cell concentration = Average cells for 1 small boxes                                                                                                       Volume of 1 small box

           with dilution aspects included the cell concentration is calculated. 

Conclusion

The cell concentration of yeast is 2.32 x 10⁷ cells per mL. By using the Neubauer chamber, we can count the number of cells and hence determine the cell concentration.

References

https://en.m.wikipedia.org/wiki/Ocular_micrometer

https://en.m.wikipedia.org/wiki/Hemocytometer

Lab 1: Principles and Use of Microscope

1.1 Setting up and using the microscope

Introduction:

A microscope is an instrument used to see objects that are invisible to the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope. There are many types of microscopes. The most common use is the optical microscope (light microscope), which uses visible light and a system of lenses to magnify images of small samples.

The diagram below shows the parts of the microscope:

STRUCTURAL COMPONENTS OF A MICROSCOPE

The three basic, structural components of a compound microscope are the head, base and arm.

•           Head/Body: Houses the optical parts in the upper part of the microscope

•           Base: Supports the microscope and houses the illuminator

•           Arm: Connects to the base and supports the microscope head. It is also used to carry the   microscope.

OPTICAL COMPONENTS OF A MICROSCOPE

There are two optical systems in a compound microscope: Eyepiece Lenses and Objective Lenses:

·         Eyepiece or Ocular: The lens at the top that you look through. Typically, standard eyepieces have a magnifying power of 10x.

·         Eyepiece Tube: Holds the eyepieces in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals.

·         Objective Lenses: The primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes.

·         Nosepiece: Holds two or more objective lenses and can be rotated to easily change power.

·         Coarse focus knob: It is used to focus on the specimen. It may move either the stage or the upper part of the microscope in a relative up and down motion.  

·         Fine Focus knobs: It is the smaller round knob on the side of the microscope used to fine-tune the focus of the specimen after using the coarse adjustment knob.

·         Stage Clips: Used when there is no mechanical stage. The viewer is required to move the slide manually to view different sections of the specimen.

·         Aperture: The hole in the stage through which the base (transmitted) light reaches the stage.

·         Illuminator: The light source for a microscope, typically located in the base of the microscope.

·         Iris Diaphragm: Controls the amount of light reaching the specimen. It is located above the condenser and below the stage.

·         Condenser Focus Knob: Moves the condenser up or down to control the lighting focus on the specimen.

Magnification and resolution:

            Magnification is the ability to make small objects seem larger, such as making a microscopic organism visible. There are four magnifications in the microscope used:

4x objective X 10x eyepiece = 40x magnification

10x objective X 10x eyepiece = 100x magnification

40x objective X 10x eyepiece = 400x magnification

100x objective X 10x eyepiece = 1000x magnification

            Resolution is the ability to distinguish two objects from each other. Not to be confused with magnification, microscope resolution is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities.

Objective:

-To learn the proper way of handling and care of microscope.

-To understand the basic concept of magnification and resolution of a microscope.

Materials and reagents:

-Microscope slide and cover-slip

Procedure:

(Refer to the lab manual)

Results 

Typical Bacillus is observed under the microscope

40x magnification

 100x magnification

400x magnification

1000x maginification (oil immersion)

 Discussion

We observed the specimen from lowest magnification that is 40x to the highest magnification 1000x (oil immersion). The specimen that we observed is Typical Bacillus. Bacillus is a genus of Gram-positive which appeared pink in color when observed under the microscope. Bacillus is one of the best understood prokaryotes, in terms of molecular and cellular biology. The cell wall of Bacillus is a structure on the outside of the cell that forms the second barrier between the bacterium and the environment and at the same time maintains the rod shape and withstands the pressure generated by the cell’s turgor. The cells are straight, round-ended or square-ended rods. The size of Typical Bacillus is tiny and the surface as it was observed under the magnification of 1000x with oil immersion, it looked smooth and the texture is moist. Most species motile by peritrichous flagella. Many Bacillus species have little or no pathogenicity and are rarely associated with disease in humans or lower animals except Bacillus anthracis, Bacillus cereus and Bacillus subtilis. Some species are insect pathogens.

Conclusion

In conclusion, we have learnt the right way of handling the microscope with care and also how to observe a specimen under the microscope from the lowest magnification to the highest magnification using a correct way. We find that the higher the magnification, the clearer the image that we can see under the microscope.

1.2 Examination of cells


Introduction

Living microorganisms are very diverse in their type, size and shape but most importantly many of them are also colorless when viewed using the microscope. It is essential as a biology student that we can search and found easily the microorganisms that is used so that we can examine and learn from it.

As such, by using the wet mount methods which does not require any special equipment, not harmful when done properly and significantly quick and easy we can studies more on microorganism easier. The wet mount methods are a technique that enable us to experience the proper use of microscope and aseptically prepare a slide thus, we are learning in a safe environment.

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 
-A microscope slide containing stained microorganisms
-Inoculating loop
-Bunsen burner 
-Slide and coverslip

Result

Lactobacillus (1000x magnification with oil immersion)

Discussion

The wet mount methods enable us to observe the Lactobacillus more clearly of the sizes and the shape. Oil immersion fills the space between the objective lens and specimen and matches the refractive index of the glass cover slip and glass objective lens. At a given focal length, greater numerical aperture can be achieved.

Lactobacillus is a genus of Gram-positive facultative anaerobic or rod-shaped bacteria. They are a major part of the lactic acid bacteria group. In humans they are part of the vaginal microbiota. Lactobacillus is a type of bacteria with multiple different species in the genus. Most Lactobacillus species in humans are considered harmless. Lactobacilli live in the urinary digestive and genital tracks of humans.Some Lactobacillus species are used as starter culture in industry for controlled fermentation in the production of yogurt, cheese, beer, wine etc.

In this experiment, aseptic techniques is important to ensure that the bacteria is not contaminated. It is also to protect the user from infection and to prevent the spread of microorganisms. Through this experiment, Lactobacillus appeared to be translucent because it was not stained.

Conclusion

The bacteria can be seen more clearly with the highest magnification under oil immersion. Added with wet mount method, the bacteria can be seen clearly in its natural state. Proper aseptic technique is important because some of the pathogenic agents can cause infection or serious illness to the user if they are not handled in a proper way. 

References

https://en.wikipedia.org/wiki/Microscope

http://www.microscope.com/education-center/microscopes-101/compound-microscope-parts/

http://sciencelearn.org.nz/Contexts/Exploring-with-Microscopes/Science-Ideas-and-Concepts/Magnification-and-resolution

www.catalog.hardydiagnostics.com/cp_prod/content/hugo/Bacillus

www.ibg102usm.blogspot.my/2014/11/lab-1-principles-and-use-of-microscope.html?m=1

www.usmibi102labreport.wordpress.com

www.en.m.wikipedia.org/wiki/bacillus.com

https://en.m.wikipedia.org/wiki/lactobacillus