PRACTICAL 2 I.

EXAMINATION OF CULTURES FOR MOTILITY

Introduction: The motility of bacteria is determined by the presence of flagella. Flagella are a long thread like structures. We can classify the organism by their motility. There are two types of determining motility, hanging drop method and soft agar stab Objective – – To identify and differentiate between true motility and Brownian motion To familiarize and perfect students aseptic techniques in handling aseptic transfers and isolating a pure culture.

Materials Per pair of students 4 soft agar bijous 2 cavity slides 2 wire loops 2 straight wires Cover slips Per bench 1 broth culture of Pseudomonas aerugenosa 1 broth culture of Staphylococcus aureus Plasticine

Procedure (A) Hanging Drop preparation 1. A tiny amount of plasticine was placed at each corner of the clean cover slip 2. One or two loopfuls of the broth culture was placed in the centre of the cover slip. 3. Concave area of the cavity was placed over the centre of the cover slip. The slip was press down gently and was inverted. 4. Under the low power objective and reducing light, the edge of the drop was centred 5. The movement of cells was observed. The movement of true motility and Brownian motion was differentiated. (A) Soft agar ‘stab’ 1. A straight wire was dip into the broth culture by using antiseptic technique. 2. One single stab was made in the centre of the soft agar down to the bottom. 3. The soft agar was not disturbed, shacked and was placed upright for incubation

I.

CULTURE TECHNIQUE

Introduction: As most microorganisms are potentially pathogenic it is essential that they be handled by procedures which avoid contamination of the microbiologist. Furthermore, since microorganisms are so ubiquitous it is necessary to avoid contamination of microbial culture by stray microorganisms. Objective – To become familiar with the basic movements of aseptic culture technique and our own technique.

1) Inoculums transfer (A) Technique of transferring culture from one agar plate to another by streaking

Materials 1 wire loop Plate culture of Escherichia coli 1 nutrient agar plate Bunsen burner Procedure Remember that the agar is a fragile medium and only gentile pressure with the oop is required.

1. The Bunsen burner was lighted and the wire loop was holed by right hand. 2. The wire loop is then sterilized by flaming it in the Bunsen burner until it is red heat. The wire loop was allowed for 5 seconds to cool it down. 3. The lid of Petri dish containing E.coli was opened by using left hand. Then, small loop was removed from the inoculums and the lid was closed immediately. 4. The second Petri dish was opened with left hand and the inoculum was spread at the small area at the top edge of the agar and spread it over a small area (primary inoculums). Then the loop was flamed. 5. After the loop was cooled make four or five single strokes starting from the primary inoculums. The loop was flamed again and overlapping series of stroke covering anther area was made until all the surface of the agar was covered. 6. The plate was closed and was placed inverted for incubation at 37’C for 24-48 hours.

(A) Technique of transferring culture from an agar plate to a bottle of nutrient broth

Materials 1 wire loop Plate culture of Escherichia coli 1 bottle of nutrient broth Bunsen burner

Procedure 1. The wire loop was sterilized and small loop was removed from the inoculum in the plate (as in 1) a) 3.) 2. The bottle was taking by using left hand and twist the cover off the bottle by using right hand (while holding the wire loop) and the top of the bottle was flamed evenly near the Bunsen burner. 3. With the right hand, the loop was dip in to the nutrient broth and was mix gently with minimum movement. 4. The bottle was flamed as in step 3 and its cap was screwed back. 5. The loop was carefully flamed by placing in the cooler part of the Bunsen flame and gradually drawn to the hotter region of the flame. (to reduce amount of splattering). 6. The bottle was labelled and incubated at 37’C for 24-48 hours

(A) Isolating a pure culture from a mixed culture

Materials A plate of mixed culture of Bacillus subtilis and Staphylococcus aureus 1 wire loop A plate of nutrient agar 1 bottle of nutrient broth

Procedure 1. The mix culture was streaked on the nutrient agar plate to obtained a pure culture of Bacillus subtilis and Staphylococcus aureus 2. The mixed culture was transferred into the bottle of nnutrient broth 3. The bottle and plate was labelled and incubated at 37’C for 24-48 hours.

Discussion: 1. EXAMINATION OF CULTUER FOR MOTILITY

Motile bacteria can move using flagella, bacterial gliding, twitching motility or changes of buoyancy. In twitching motility, bacterial use their pili as a grappling hook, repeatedly extending it, anchoring it and then retracting it with remarkable force (>80 pN). Bacterial species differ in the number and arrangement of flagella on their surface, some have a single flagellum (monotrichous), a flagellum at each end (amphitrichous), clusters of flagella at the poles of the cell (lophotrichous), while others have flagella distributed over the entire surface of the cell (peritrichous). a) Brownian movement is caused by the molecules of the suspending liquid colliding with the organism and moving it around in an irregular, jerky pattern. This is not considered to be true motility. While on the other hand true motility's movement in some consistent direction or path, even though there may be many twists and turns. The result are: Staphylococcus aureus - non-motile cocci Pseudomonas aeruginosa- unipolar motility

Staphylococcus aureus Pseudomonas aeruginosa

The hanging drop method has several advantages and disadvantages: Advantages: -The hanging drop method allows the organism to live longer because it does not dry out as quickly. -The hanging drop method also offers a better view of true motility.

-The Vaseline-sealed depression or sometimes using plasticine,also slows down the drying-out process, so the organisms can be observed for longer periods. Disadvantages: -The hanging drop method is also far too risky to use with highly pathogenic organisms.Human will get infection easily.

b) Motility Test medium Semi-solid Motility Test medium may also be used to detect motility. When a non-motile organism is stabbed into Motility Test medium, growth occurs only along the line of inoculation. Growth along the stab line is very sharp and defined . When motile organisms are stabbed into the soft agar, they swim away from the stab line. Growth occurs throughout the tube rather than being concentrated along the line of inoculation. Growth along the stab line appears much more cloud-like as it moves away from the stab. If a tetrazolium salt (TTC) is incorporated into the medium. Bacterial metabolism reduces the TTC producing formazan which is red in color. The more bacteria present at any location, the darker red the growth appears.

Pseu. Aeruginosa

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Staph. aureus

However no color indicator used in this experiment.The advantages and the disadvantages are: Advantages: This technique is useful for determining the motility of highly pathogenic bacteria. It is also easier to read than the other techniques. Disadvantages: This technique takes longer to provide results than the wet mount or hanging drop.

1.

CULTURE TECHNIQUE

Cell culture is the process by which cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. 1) Inoculums transfer a) Technique transferring culture from one agar plate to another by streaking. Culturing bacteria using broth culture is a good technique for determining pure culture.

E. coli in agar medium The advantages and disadvantages of this technique are: Advantages: -Growth of bacteria on solid media has advantages over use broth cultures because it allows isolation of bacteria in pure culture. It is because a colony well separated from others can be assumed to rise from a single organism cluster (CFU). Disadvantages: -Anaerobic bacteria will not grow. - It does not always provide the clear separation of inoculums / bacteria desired. - Also a chance of carry-over and contamination from one portion to another. - Additionally, it is often difficult to determine what portion of the sphere has already been used. - Essentially a storage place for your bacteria.

b) Technique transferring culture from one agar plate to a bottle of nutrient broth. Culturing bacteria using broth culture is a easy and good technique.

E. coli in broth medium The advantages and disadvantages of this technique are: Advantages: -Allows for rapid and large volume of growth bacteria. - It suitable for long-term storage. Disadvantages: -We cannot count bacterial colony directly, it must use another method such as miles-misra method, pour plate method, most probable number method, or other method to isolate and count colony formed. 2) Isolating a pure culture from mixed culture Part 1 a. Observe and sketch the distribution of growth:

Mix Culture

b. No. It is because I do not streak the culture properly. Maybe third or fourth streak had touch first streak. Dilution process cannot occur. Cannot form isolated colonies. But the colonies same with original plate. c. Broth Culture:

Before After Before inoculate it is clear, after inocculate it become cloudy or turbid.It is because present microorganism in the broth medium. Less light penetrate the broth medium because most of light had been absorbed or reflect by the microorganism. d. Definitely no, because we cannot determine the separated colonies in the medium. The colonies mixed evenly in broth medium. Part 2 a. I am not able to separate the 2 organisms successfully. It is because I am late to observe it. We observed after 4 days although I must observe after 1 day. It also due to error during streaking process. Maybe I had touched the first streak during forth streak. Diluting process failed. Cannot form pure culture colony. Mix culture: Shape-irregular and filamentous Texture - smooth Color - white colony Elevation - raised from medium surface E. coli : Shape-irregular Texture - smooth Color - white colony

Elevation - raised from medium surface
b. When the culture is being contaminated, we can see many different

colony occur that determined by different color and shape. c. We incubate the inoculated in inverted position because to prevent culture from being contaminated easily.
Refferences: : -Tortora, Funke, Case’s Microbiology: An introduction. -Levinson & Jawetz’s Medical Microbiology & Imunology. -www.answers.com -www.wikipedia.com -http://in.answers.yahoo.com/question/index? qid=20061024232001AAP0ikl

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