STREAKING METHODS

 

STREAKING METHODS

INTRODUCTION:

Streaking is a common method used to isolate a particular bacterial colony from a mixture of bacteria that is pure culture. In the streak plate method, the concentration of bacterial colonies is more at the starting point and it goes on decrease toward the last point of the streak. It helps to isolate individual colonies from other colonies and each colony is considered a pure colony.

During the identification of a microorganism, the first and important step is to isolate the individual species from a mixed sample. This is mainly done by the streak plate method; therefore streak plate method is an isolation technique.

In this method a sterile inoculating loop is first dipped into a diluted bacterial culture; then the culture-containing loop is streaked on the surface of a solidified agar plate to make a series of parallel, non-overlapping streaks.

As the culture is diluted before streaking on solid agar, the organism number will decrease by the third or fourth quadrant. Therefore only a few bacterial cells are transferred on the solidified agar medium as a result it will give discrete colony forming units (CFUs).

During the streaking an agar plate different patterns are used, depends on the source of inoculum and the microbiologist’s preference. The patterns of streaking patterns range from simple to more complex, these are designed to separate deposited cells (CFUs) on the agar surface so individual cells (CFUs) grow into isolated colonies.

A quadrant streak pattern is used for samples suspected of high cell density, while a simple zigzag pattern used for samples containing lower cell densities.

AIM: To perform the Streak plate method on different media

REQUIREMENT:

• Bacterial culture: 24 hours bacterial culture
• Apparatus: Sterile Petri dishes, inoculating nichrome wire loop, burner, marking pen.
• Media: Nutrient agar media.
• Equipments: Autoclave, Colony Counter, Hot air Oven, Incubator.

General Procedure:

1. Petri dish is labeled on the bottom.
2. Sterilize the nichrome wire loop on the flame of the bunsen burner.
3. Open the bacterial culture tube and collect a sample of bacterial culture with the help of a sterile nichrome wire loop.
4. Streak the nutrient agar plate with bacterial culture. The lid of the agar plate has to be opened between the bunsen burner and streak out the labeled quadrants.
5. Make sure that incinerate the loop before and after inoculating the bacterial culture.
6. All the process is done in a strictly aseptic condition in a laminar airflow cabinet.

Three Sector Streak (t- streak) & Zig - Zag Streak Method:

1. Sterilize the nichrome wire loop on the flame of a bunsen burner.
2. Cool the wire loop between the bunsen burner.
3. Dip the wire loop into the broth culture containing the mixture of bacteria.
4. Insert the nichrome wire loop on the nutrient agar plate and streak the bacterial suspension in a zigzag manner and forms T-shaped streaking.
5. Incubate the plate for 24 hours and you will see isolated colonies in the third sector. There will be less growth in the second sector and the heaviest growth in the first sector.

Four Quadrant Streak method:

1. Sterilize the Nichrome wire loop on the flame of a bunsen burner.
2. Cool the wire loop between the burner.
3. Label the petri dish with a marker and mark four-quadrant on the base of the petri dish.
4. Flame the test tube which contains bacterial culture.
5. Insert the nichrome wire loop into the culture tube.
6. Streak the bacterial suspension in the four-quadrant of the plate between the two burners.
7. Incubate plate at 37°C for 24 hours.

 

Observation: Examine the growth of isolated colonies on the surface of the nutrient agar plate.

Result: Few numbers of isolated colonies appear along with the points of the streak.

STERILIZATION AND MEDIA PREPARATION

 

STERILIZATION AND MEDIA PREPARATION

 

 Introduction:

 

Sterilization: Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121 degree Celsius 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 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121 degree Celsius. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 degree Celsius throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilized objects can then be removed.

Media Preparation: Microorganisms depend on a number of factors such as nutrients, oxygen, moisture and temperature to grow and divide. In the laboratory, except for the above factors, the culture medium should be sterile and contamination of a culture with other organisms should be prevented.

The properties of agar which make it ideal in bacteriology are

1) Solid agar melts (dissolves) at 100 °C,

2) Remains solid at all incubation temperatures,

3) Transparent,

4) It is not heat-labile and therefore easily sterilized, and

 5) It is unaffected by almost all bacteria.

Liquid agar solidifies at 42-44 °C which is useful because sterile, heat-labile components such as antibiotics, blood, serum, carbohydrates and even bacterial cultures may be added before allowing the medium to solidify. Solid media generally contain agar at a concentration of 1.5%. Semi-solid media contain 0.05-0.3% agar and are useful in culturing anaerobic and microaerophilic organisms because such media form an oxygen gradient in test tubes, allowing all degrees of oxygen tension to exist in the culture vessels.

AIM:

            To understand the sterilization process using an autoclave and to learn the procedures used in preparing media needed for culturing microorganisms.

MATERIALS REQUIREMENT:

 

Commercial nutrient agar, Balance, Distilled water, Scott bottles, Measuring cylinder Beaker, Forceps, Universal bottles

PROCEDURE: STERILIZATION

The usual procedure for sterilization using the autoclave is as follows:

1. Open door, and place items to be sterilized into the autoclave chamber.

2. Close door. Push down door lock lever until door studs are completely in place.

3. Turn hatch wheel clockwise until it is secured tightly.

4. The temperature of the autoclave is set at 121°C.

5. Set timer by turning the large knob just below the hands to the desired setting.

6. Crank operating handle around to the Sterilize position till the red steam light goes on.

7. Wait and be sure the chamber reaches the proper temperature and pressure.

9. The Slow Exhaust and Fast Exhaust & Dry cycles both take 12-15 minutes longer than the time set to finish.

10. At the end of the run the white STERILE light will go on, and a loud, obnoxious buzzer will come on:

a. Rotate the operating handle all the way to OFF. Check that the chamber pressure is zero, and the temperature is below 100 °C.

b. Turn hatch wheel counterclockwise, push up door lock lever and slowly open door.

c. Use heatproof gloves to remove materials.

11. Allow liquid materials to cool before tightening caps.

PROCEDURE MEDIA PREPARATION:

 

1.      Measure approximately 250 ml of distilled water in a 1 L graduated cylinder and pour into a 1 L flask.

2.      Weigh out 1.5 g beef extract and 2.5 g peptone and add into the flask. Use approximately 100 ml of the water to rinse any powder stuck to the side of the flask down into the mixture.

3.      Stir over gentle heat from a bunsen burner to dissolve completely.

4.      Pour the mixture into the 1 L graduated cylinder and add warm water to the 500 ml mark. Pour back into the flask.

5.      Check the pH of the medium and adjust to pH 7.

6.      Autoclave the flask and the petriplates for 15 minutes at 121 °C and 15 lb/in2 pressure at the slow exhaust mode.

7.      After removing the media from the autoclave, allow the media to cool, and store for later use.

8.      Lay your petri dishes on the bench. The cover should be on top. Light your Bunsen burner, and then remove the NA flask from the water bath.

9.      Remove the tapes and cotton plug from the flask. Carefully flame the neck of the flask, open the plate cover about half way and pour the media in to petri plate about 30 ml.

10.  Flame the neck of the flask between each plate. Allow plates to solidify completely, which will take 15 minutes. Then invert, label and incubate at 37 °C overnight to dry off excess moisture and check for contamination.

 

CONCLUSION:

Different types of agar are needed for the cultivation of different types of microorganisms. Agar of the same composition with the commercial agar can be made by following the correct procedures. Preparation and sterilization of culture media should be done with great care to avoid contamination of unwanted microorganisms. We had learnt the preparation and sterilization of culture media via autoclaving process and the precaution that we need to take into consideration when handling this experiment.

 

PREPARATION OF CLEANING SOLUTION: ETHANOL

 

PREPARATION OF CLEANING SOLUTION: ETHANOL

INTRODUCTION

Ethanol is a flammable chemical. If not stored and handled properly, this can pose a serious threat to the health and safety of laboratory personnel, emergency responders and chemical waste handlers. Hence, it is important to follow safety protocols to handle this chemical. Ethanol is mainly used as solvent. It is miscible with water and with many organic solvents, including acetic acid, acetone, benzene, carbon tetrachloride, chloroform, diethyl ether, ethylene glycol, glycerol, nitromethane, pyridine, and toluene. It is also miscible with light aliphatic hydrocarbons, such as pentane and hexane, and with chlorinated organic such as trichloroethane and tetrachloroethylene.

AIM:

To prepare the cleaning solution for disinfect the surface of working table and glassware.

MATERIALS REQUIRED:

            Measuring jar, 0.2 micron filter, Distilled water, 99.9% ethanol, Filtration apparatus, Glass bottle

PROCEDURE:

1.      Assemble the filtration unit to achieve filter sterilization to the water and the 99.9% lab ethanol. All the steps shall be performed in to the LAF to avoid contamination.

2.      Pour the water in to the 0.2 micron filter containing filtration cup and turn on the pumb.

3.      Repeat the procedure for sterilizing the lab ethanol.

4.      To prepare 1 liter of 70% Ethanol using 95% lab Ethanol add 740ml of lab ethanol to 260ml water.

5.      Label the container and store in a safe place.

RESULT:

Ethanol cleaning solution has been prepared as followed the procedure for the disinfection purpose and stored in a secure place. The effectiveness of the cleaning solution will be checked periodically.

 

SAFETY IN MICROBIOLOGY LABORATORY

 

Safety precautions in the microbiology laboratory:

Specific instructions that should be followed in the microbiology laboratory:

1. If you have any Illness, should share with your lab instructor.
2. Wear gloves when working with cultures, and when your work is completed, dispose of the gloves in the biohazard garbage. Lab coats, safety glasses or goggles are also required. 
3. Disinfect the work area both BEFORE and AFTER working with bacterial cultures.
4. Cultures of live microorganisms and any material coming in contact with live cultures must be properly sterilized after use in the laboratory.

5.      Glassware such as test tubes, bottles, and flasks may be reused and washed after sterilization. These are normally placed on a cart at the front of the laboratory after you have finished an experiment or exercise. BE SURE TO REMOVE LABELS before placing any glassware on the cart.

6.      Materials, such as plastic petri dishes, plastic pipettes, microscope slides,
and swabs, are considered disposable. These are used once and if they become
contaminated by contact with live microorganisms are sterilized and discarded.
All of these disposable contaminated materials should be placed in the designated
waste container containing a BIOHAZARD autoclave bag.

7.      Never place contaminated pipette tips (or pipettes), inoculating loop, or any other contaminated material on the bench top.

8.      Sterilize loops before and after each use. Do not place or put anything containing live microorganisms in the sink.

9.      Aerosols should be avoided by the use of proper technique for sterilizing the inoculating loops and by performing any mixing of cultures and reagents in such a way as to avoid splashing.

10.  Cultures or reagents should always be transferred with an automatic pipettor that will be provided. In no case should one employ mouth pipetting.

11.  Always keep cultures capped and in proper storage racks when not being used during an exercise.

12.  In case of an accidental spill of a bacterial culture, completely saturate the spill area with disinfectant, then cover with paper towels and allow the spill to sit for 10 minutes. Then carefully remove the saturated paper towels, dispose of them in the biohazard waste, and clean the area again with disinfectant.

13.  All accidents and incidents shall be reported and documented such as spills, cuts, burns, or other injuries to the instructor

14.  Make sure that lab benches are completely cleared before you leave the lab.

15.  Clean and washed Clothing worn in the microbiology laboratory.

 

BIOHAZARD SYMBOL