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

DETERMINATION OF MIC

 

Determination of Minimal Inhibitory Concentration

AIM: To identify the MIC of given antimicrobial compound or antibiotic stock solution.

PRINCIPLE: The agar dilution technique is used to measure qualitatively the in vitro activity of an antimicrobial agent against the test bacteria. In this method, graded amounts of antibiotics are incorporated in agar plates and inoculated in spots with the organisms under study. If the organism under study is susceptible to the incorporated antibiotic, no bacterial growth is expected in agar plates with higher amounts of the drugs. Bacterial growth is observed as the antibiotic concentration in the agar plate diminishes. Inhibition of growth at the minimum or lowest concentration of antibiotic is regarded as the end point.

MATERIALS REQUIRED: Culture media, solvents, antimicrobial agents, control strains and apparatus needed for the minimal inhibitory concentration (MIC) test.

PROCEDURE:

1.      Dissolve the antimicrobial agent powder in solvent to make a concentration of 1,000 µg/ml.

2.      Dispense the stock solution into sterile diluents using two-fold dilution technique.

3.      Prepare MHA and Keep in a water bath at 48-50°C until use.

4.      Label each empty sterile plate in order to identify the antimicrobial agent and their concentrations.

5.      Pipette 1 ml of appropriate dilutions of the test antimicrobial agent into the labeled plate. Two replicates must be made for each concentration.

6.      Pipette 9 ml of MHA (keep warm at 48- 50°C), add into the plate with appropriate dilution of the test antimicrobial agent and mix thoroughly.

7.      Allow the agar to solidify at room temperature and Control agar plates/Drug-free agar plates

8.      Do not add any antimicrobial agent. There should at least be 2 control plates.

9.      From a pure 18-24 hour bacterial culture get 4- 5 isolated colonies.

10.  Shake vigorously in a water bath at 30°C until it achieves or exceeds the turbidity of 0.5 MacFarland standard (prepared by adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.36 NH2 SO4 ; commercially available). The inoculum may also be standardized based on optical density [OD625 of 0.08-0.1 (1cm light path)] using a spectrophotometer. This is usually achieved after 18- 24 hours.

11.  Dilute the standardized inoculum 1:10 in sterile saline solution to obtain the desired concentration of 10⁶ cfu/ml.

12.  Pipette 0.1 ml of the 10⁶ cfu/ml inoculum and transfer to a well, sterile test tubes of the same size, may be used to hold the diluted standardized inoculum.

13.  Inoculate plates with 10 µl.

14.  When inoculating manually, it is only important to include a drug free or control plate at the beginning of the inoculation series.

15.  Inoculate the bacterial suspensions onto the surface of the agar plate.

INCUBATION

16.   Incubate the plates in an inverted position at 30°C for 18-24 hours.

17.  Read and record the MIC at the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the naked eye.

18.  Report result as Resistant (R), Intermediate (I) or Susceptible (S).

Example: Antibiotic: Oxytetracycline MIC breakpoint: 0.2 µg/ml Interpretation: susceptible.

Stock Solution Preparation

a. At least 1,000 µg/ml or 10 times the highest concentration to be tested is to be prepared as an antimicrobial agent stock solution.

Some antimicrobial agents are of limited solubility. Therefore, lower concentration may be required.

 b. Some drugs must be dissolved in solvents other than water.

Prepare stock solutions using formula

1000  xVxC=W    P

 

  

P-potency (µg/mg), V-Volume required (ml), C-Final concentration of solution (mg/L), W-Wt of antibiotics dissolved in volume V (mL).

 

CONJUGATION

 

CONJUGATION

AIM:

To study the process of bacterial conjugation through transfer of genes coding for antibiotic resistance.

PRINCIPLE:

Conjugation is the mode of gene transfer in many species of bacteria. In 1950 William Hayes, Francis Jacob and Elie L. Wollman established that conjugating bacteria are of two mating types. Certain “male” types (designated as F+) donate their DNA and other “female types” (designated as F-) receive the DNA. F- cells become F+ when they acquire a small amount of DNA. Hence the F factor is called as the Fertility factor. In contemporary microbiology, the donor’s F factors are known to be plasmids which are the extrachromosomal elements. The factors (plasmids) contain about 20-30 genes, most of which are associated with conjugation. These genes encode enzymes that replicate DNA during conjugation and structural proteins needed to synthesize special pili at the cell surface. Known as F pili or sex pili, these hair like fibres contact the recipient bacteria, and then retract so that the surfaces of donor and recipient are very close or touching one another. At the area of contact, a channel or conjugation bridge is formed. Once contact via sex pili has been made, the F factor (plasmid) begins replicating by the rolling circle mechanism. A single strand of the factor then passes over through the channel to the recipient. When it arrives, enzymes synthesize a complementary strand, and a double helix is formed. The double helix bends to a loop and reforms an F factor (plasmid), thereby completing the conversion of recipient from F- cell to F+ cell. Meanwhile, back in the donor cell a new strand of DNA forms, to complement the leftover strand of the F plasmid. The transfer of F factors involves no activity of the bacterial chromosome; therefore the recipient does not acquire new genes other than those on the F factor.

MATERIALS REQUIRED:

Glass wares: Conical flask, Measuring cylinder, Sterile test tubes, Petri plates

Reagents: Distilled water

Other requirements: Incubator, Shaker, Spectrophotometer, Micropipettes, Tips, Sterile loops and spreaders

PROCEDURE:

MATERIALS

1X PBS (Phosphate-buffered saline )

Lysogeny agar

Lysogeny

STEP MATERIALS

1X PBS (Phosphate-buffered saline )

1X PBS (Phosphate-buffered saline )

PROCEDURE:

Day 1:

1.      Open the vials containing Donor and Recipient cultures and resuspend the cells with 0.25 ml of LB broth respectively.

2.      Pick up a loopful of Donor culture and streak onto LB plates with Tetracycline (30 μg/ml).

3.      Pick up a loopful of Recipient culture and streak onto LB plates with Streptomycin (100 μg/ml). 4. Incubate overnight at 370 C.

Day 2:

1.      Pick up a single colony from Donor and Recipient Strain grown overnight on LB plates and inoculate a single colony in 6 ml of LB broth having respective antibiotics.

2.      Incubate the test tubes overnight at 37o C.

Day 3:

1.      Take 25 ml of LB broth and add 25 μl of tetracycline into it and inoculate 1 ml of overnight grown culture into it. Incubate at 37oC in a shaker.

2.        Take 25 ml of LB broth with streptomycin at a concentration of 100 μg/ml and inoculate 3 ml of overnight grown culture in it. Incubate at 37oC in a shaker.

3.      Grow the cultures till O.D of the donor culture reaches 0.8-0.9 at A600.

4.      Add 0.2 ml of each donor and recipient cultures in a sterile test tube labeled as conjugated sample. Mix by gentle pipetting and incubate at 37oC for 1-1.5 hours.

5.      Take 2 sterile test tubes and label them as donor and recipient. Add 0.2 ml of respective cultures to the test tubes and incubate at 37oC for 1-1.5 hours.

6.      Add 2 ml of LB broth into each tube after incubation. Incubate the tubes at 37oC for 1.5 hours.

7.      Plate 0.1 ml of each culture on the antibiotic plates as indicated in Table .

8.      Incubate the plates overnight at 37oC overnight.

Observation and Result:

 

	LB + (Streptomycin)+X gal	LB + (Tetracycline)+IPTG	LB + (Streptomycin, Tetracycline)+X gal+ IPTG
Donor Strain A
Recipient Strain B
Conjugated Sample

NOTE: Keep uninoculated LB plate as control.

Interpretation:

On observing colonies on different plates the following interpretation can be made:

1.        Donor strains will grow only on tetracycline plates, similarly recipient strains will grow only on streptomycin plates.

2.        Donor strain is sensitive to streptomycin and recipient strain is sensitive to tetracycline, hence no growth will be seen in these plates.

3.        The conjugated sample will grow on tetracycline and streptomycin plate. The reason being, transfer of gene has occurred by means of conjugation.

4.        The donor and recipient strain will not grow on tetracycline + streptomycin plate since each of the strain is sensitive to one antibiotic in the plate.