Needle Aspirations To Collect Blood Biology Essay

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Techniques used on obtaining specimens: This largely depends on what type of illness the animal obtained. Many laboratories that are specific for disease agents that are controlled need adequate clinical history to accompany the submissions. Samples can be obtained from dead or live animals, but is preferred to be live, sick animals taking the suspected pathogens and phase of disease into account. In live animals nasal or ocular swabs, vesicular fluid, faeces or faecal swabs, clotted and unclotted blood can all be collected for examination. In animals that are dead it must be kept in mind to collect samples as soon as possible after death. Recommended tissues for examination include lung, kidney, liver, spleen, small intertine, large intestine, and lymph nodes. Brain tissue and head should be collected if a disease is suspected from the central nervous system. Blood samples should then also be collected. The following can be collected:

Sterile swabs

Needle aspirations to collect blood and cerebrospinal fluid; anticoagulants are used to prevent blood clotting

Intubation used for samples from the stomach

Catheterisation used for the collection of urine samples

Clean catch midstream urine

Sputum - mucous secretions from lungs, bronchi and trachea

Preventative measures: for blood samples it must be collected and placed into anticoagulant tubes such as Heparin and EDTA. Never leave your samples in the heat and also never freeze them. Always keep your samples refrigerated.

The samples must reach the laboratory as soon as possible, especially for controlled animal diseases. Samples can be curried via aeroplane, bus and using currier services, but must never be transported on train. Laboratory staff must use the standard microbiological practices when handling specimens so that no harm will come to them.

Specimen preservation: each type of specimen has a different type of preservative that it should be placed in. Like the blood it must be placed into anticoagulant tubes. Abortions must be in a cool place and submitted in a watertight container. Aerobic bacterial cultures: swabs in transport medium and tissues in separate sterile containers. Anaerobic bacterial cultures - sterile containers; keep cool and rush. Slides of smears must be wrapped individually in tissue paper and packed between cardboard. Cultures must be sealed in metal containers. Cultures for typing placed in petri dishes and kept cool. Freeze or cover organs with 10% formalin in a thick sealed plastic bagor suitable container. Tissue blocks for pathology kept in 10% buffered formalin in a wide mouth jar. It really all just depends on the specimen collected.

The use of antibiotics: antibiotics will control the bacteria in using certain products by resisting specific factors which influences their growth and reproduction.

Documentation needed when collecting samples: most laboratories supply a specimen submission form where you complete the available pertinent information. In the absence of a form a veterinarian should supply a complete history. On all the samples the following information must be present: name of specie, address of farmer, telephone number of the farmer, type of specimen (e.g. brain, lungs etc.), date, and suspected pathogen and how big the herd is.

Processing the specimen

Isolation of pure cultures: this is done by using culture transfer techniques. Pipettes are used to transfer aliquots of culture, prepare serial dilutions of microorganisms, and dispensing chemical reagents. Using a pipette you can do the following transfer methods: from a broth to an agar plate and from a broth to another tube with microorganisms. You can use an inoculating loop after which you flame it first to kill the organisms on it, cool it in the media and transfer the microorganisms on the media to another type of media. You can also use an inoculating needle to do some of these transfers. Further on there is the pour plate method where you dilute a sample with sterile saline or phosphate buffer to reduce the microbes sufficiently and obtain separate colonies when plating. The streak plate method is where the bacterial mixture is transferred to the edge of your agar plate and streaked out over the surface in one of several patterns with an inoculating loop. In the spread plate method, a small volume of dilute bacterial mixture which will contain 100-200 cells or less is transferred to the centre of an agar plate and spread out evenly with a sterile, L-shaped glass rod. Remember the following when isolating pure cultures: be organized (arrange your media and label them with your name, date, medium and the microorganism to be transferred); take your time (if you do everything in a hurry, you might harm yourself with potentially dangerous microorganisms); place all media tubes in a test tube rack when not in use, hold the handle of the inoculating loop or needle like a pencil; adjust the Bunsen burner; hold the culture tube in your non-dominant hand; grasp the tube's cap with your little finger; flame your tube passing it through the Bunsen burner two or three times; hold open tubes at an angle (this will minimize the chance of airborne contamination); suspend bacteria in broth with a vortex mixture prior to transfer; and when opening a plate, use the lid as a shield to minimize the chances of airborne contamination.

Selective media: this is media that allows only certain types of organisms to grow in or on them because of 1) absence of certain nutrients that make it unfavourable for the microorganisms 2) the presence of inhibitory substances that prevent growth of microorganisms. This includes salt (NaCl), acid, a toxic chemical (crystal violet), an antibiotic (streptomycin) or other substances.

Differential media: contain substances that cause some bacteria to take on a different appearance from other species. In this manner one can differentiate one specie from another. Media that are both selective and differential have been formulates to determine the presence of coliforms in water analysis, such as Levine EMB agar.

Media to determine biochemical reactions: used to test bacteria for particular metabolic activities, products and requirements. These are for example: urea broth to detect the enzyme urease; triple sugar iron agar(TSI) used to identify enteric organisms that attack glucose, lactose, or sucrose and that liberate sulphides; citrate agar used to differentiate enteric bacteria based on their citrate utilization; Lysine iron agar (LIA) which can be differentiated by bacteria that can either deaminate or decarboxylate the amino acid lysine; sulphide indole motility (SIM) is used to observe the production of sulphides, the formation of indole, and motility.

Plating techniques

Streak plate

Principle:

This technique was developed by Fredrich Loeffler and George Gaffky. During this technique, bacterial cells from the original sample will be spaced apart from each other and the numbers will reduce tremendously. The plate is incubated and allowed for bacteria to grow.

Materials:

Unknown organism

sterile petri plates

Permanent marker

48 to 50 water bath

Bunsen burner

Inoculating loop

Method:

Prepare media of choice and sterilize the medium in the autoclave

Cool the media for about 10 minutes in a 48 to 50 water bath

Remove the cap of the bottle containing the media, flame the top of the bottle and pour the agar into the petri plates keeping the lid of the petri plate just slightly above the bottle

Allow the petri plates to solidify for about 20 minutesby leaving them on the bench for a few minutes. When solidified mark the bottom of the petri plates with organism number (which is number 17), your name and date

Remove a loopful of bacterial mixture aseptically and streak it out on the agar plates as follows:

Remove the lid of the petri plate preventing airborne contamination. Insert the inoculating loop consisting with the bacteria and spread it over a small area. Do this by gently letting the loop rest on the agar and moving it across the plate preventing digging into the agar

Remove the inoculating loop and flame it. Insert the loop and cool it in the agar

From the first area, rotate the plate and streak out a second area.

Remove the inoculating loop and flame it. Streak out the third and fourth area using the same process.

Repeat the procedure if there are more than one petri plates used

Incubate for 24-48 hours in an inverted position at 30. Examine each of the agar plates for results

Results:

Growth of organisms are found on Tryptic soy agar (TSA) agar in a white colour and exactly where streaks were made

Discussion and conclusion:

Growth on TSA media is found, but I will have to make more streak plates on other types of media to isolate and determine what organism I have.

Pour plate

Principle:

We must isolate pure cultures to be able to study the cultural, morphological and physiological properties of the specie. It consists of diluting samples with a sterile saline reducing the microbial population for sufficient separate colonies. Fewer than 25 and more than 250 colonies are plated due to the fact that the exact number of bacteria in a sample is unknown.

Materials:

Unknown organism

3 tryptic soy agar pour tubes

9ml sterile saline 0,9% NaCl (saline) blanks (x3)

48 to 50 water bath

Permanent marker

3 petri plates

Inoculating loop

Bunsen burner

3 sterile 1 ml pipettes with pipettor

Method:

With a permanent marker, mark 3 sterile saline blank tubes 1-3

In a 48 to 50 water bath meth the tryptic soy agar deeps for about 10 - 15 minutes

Mark the 3 petri dishes numbering them 1-3, add your name date and organism number with a permanent marker

Inoculate saline tubes with 1 ml of the unknown organism keeping the aseptic technique in mind. Mix thoroughly. This is referred to as a 10-1 dilution

Inoculate the second tube immediately with 1 ml aliquot from tube 1 making a 10-2 dilution

Mix the contents of the second tube and use it to inoculate the third tube with 1 ml from tube two, to finally make a 10-3 dilution. Mix the contents

Remove the tube cap, flame the top and transfer 1ml aseptically into petri plate 3. Inoculate tube 1 and 2 representatively

Add the melted tryptic soy agar pours to the petri plates and mix each of them gently in a circular motion still keeping it flat on the bench. Leave the plate to cool down and harden

Incubate the plates for 24-48 hours keeping it in an inverted position

Examine the plates and record the results

Results:

Pour plate 1: white cultures all over the petri plate

Pour plate 2: white cultures much less throughout the petri plate

Pour plate 3: white cultures all over the petri plate but in small amounts. Cultures big as needles counted at 135 colonies

Discussion and conclusion:

The cultures were correctly minimized to 135 colonies on the third petri plate. This means that the isolation of single colonies was a success and I now know that I can mathematically count how much organism i will have in my entire tube.

Spread plate

Principle:

For individual characterization of species, pure cultures must be done. This technique uses a small volume of a dilute bacterial mixture that contains about 100-200 cells or less that is transferred to the middle of the agar plate and using a L-shaped glass rod, spread the mixture over the surface evenly. The glass rod is sterilized by dipping it in alcohol and flaming it straight after. The general form and shape of the colony can be examined looking down from the top of the colony. It can further be picked up and streaked using a fresh medium that will obtain pure cultures

Materials:

Unknown organism from broth culture

Bunsen burner

Inoculating loop

L-shaped glass rod

Permanent marker

Pipettes with pipettor

Tryptic soy agar plate

Rulers

Method:

Using the permanent marker, add to the bottom of the agar medium plates your name, organism number and date

Add 0,1 ml of your unknown organism to the centre of a tryptic soy agar plate using a pipette

Pass the L-shaped rod through the flame to sterilize, cool it in the medium on the side and spread the sample evenly over the agar plate. Do not tough the edges of the plate

Reflame the L-shaped rod to sterilize it

Incubate the plates for 24-48 hours at around 30 in an inverted position

After incubation, measure the colonies and observe their morphology. Record the results

Results:

They are punctiform with a raised elevation on the entire margin. They are dull, opaque with a non-pigmented white colour and smooth.

Discussion and conclusion:

According to my results where my colonies are white, I think that my organism is not a Micrococcus specie due to the fact that they have red colonies on TSA plates.

Colony counting and characteristics of the isolated bacterium

My colonies are small in punctiform with a raised elevation on the entire margin. They are dull, opaque, smooth and non-pigmented (white). With the pour plate technique my colonies were counted as 135 colonies also in a punctiform with non-pigmented (white) colonies.

Smears and staining

Gram staining

Principle:

The Gram staining procedure was named after Christian Gram. This is the most useful and widely employed stain available. It divides bacteria into gram positive and gram negative groups. It involves staining with a basic dye such as crystal violet. It is then stained with a mordant such as grams iodine. The smear is decolorized with a 95% ethanol or propanol acetone. Gram positive bacteria will retain the primary stain, whereas gram negative bacteria will become colorless. It is finally stained with a counterstain such as safranin. The gram negative bacteria will then stain pink/red, whereas gram positive bacteria remain purple. Gram positive cultures can become gram negative if they are too old. Other species may be gram variable.

Materials:

18-24 hour tryptic soy agar plate containing the unknown organism

Crystal violet

Grams iodine

Isopropanol acetone mixture

Inoculating loop

Immersion oil

Paper towel

Microscope

Sterile distilled water

Microscope slides

Permanent marker

Lighter

Bunsen burner

Method:

Add a drop of water to a microscope slide. Sterilize the inoculating loop putting it through the flame, cool it in the agar and take up organisms and mix it thoroughly with the drop of water on the microscope slide. Heat fix the slide

Put the slide on a staining rack

And crystal violet to the slide and keep it on for 30 seconds

Rinse off with water for 5 seconds

Add grams iodine mordant and let it stand for 1 minute

Rinse off with water for 5 seconds

Decolourization with isopropanol-acetone for 30 seconds

Rinse off with water for 5 seconds

Blot dry with a paper towel

Examine under the microscope and add immersion oil if necessary

Results:

Organisms stain purple and are cocci shaped occurring in short chains, pairs and clusters.

Discussion and conclusion:

I think that my organism according to my results will be either a Staphylococcus or Streptococcus specie due to the fact that they are gram positive cocci and the way they are arranged.

Acid-fast staining

Principle:

Organisms that cannot be stained with simple stains can however be stained by heating them covered in carbolfushin. The heat will readily drive the stain into the cells. The honour goes to Paul Erlich who developed the procedure in 1882. Acid fast organisms will readily retain this dyeto appear red. If not acid-fast, they will appear blue or brown. An e.g. of a acid-fast negative stain:

Materials:

Unknown organism on TSI agar plate

Acid alcohol

Alkaline methylene blue

Ziehl's carbolfushsin

Paper towel

Inoculating loop

Immersion oil

Microscope

Microscope slide

Permanent marker

Bunsen burner

Method:

Add a drop of water to a microscope slide. Sterilize the inoculating loop, cool it in the medium and take up organisms from the edge of the agar plate and mix it thoroughly with a drop of water on the microscope slide. Allow to air dry for about 5 minutes and then heat fix the slide

Add Ziehl's carbolfushsin to the slide and heat for 3-5 minutes. Do not allow it to dry out and avoid excess flooding. Be careful not to keep the microscope slide over the Bunsen burner too long, as the microscope slide gets too hot and breaks.

Allow the slide to cool and rinse thereafter with water for 30 seconds

Using acid-alcohol, add drop by drop until a slightly pink colour remains. This decolorizes the organisms

Rinse for 5 seconds with water

Use alkaline methylene blue to counterstain for 2 minutes

Rinse for 30 seconds with water

Blot dry the slide with paper towel

Examine the slide under the microscope and add immersion oil if necessary

Results:

Organisms and background stains blue

Discussion and conlusion:

Due to the organisms and background staining blue it means that my unknown organism is non-acid fast

Spore staining

Principle:

Spores are capable of producing resistance and surviving for larger periods in unfavourable environmental conditions and develop within the bacterial cell. They have spherical to elliptical shapes and can be either smaller or larger than the parent bacterial cell. They are difficult to stain, nut when finally stained they resist decolorization. Staining procedures are based on Schaeffer-Fulton or Wirtz-Conklin methods. E.g. of a spore stain:

Materials:

24-48 hour agar plate with unknown organism

Microscope slide

Immersion oil

Permanent marker

Inoculating loop

Malachite green solution 5%

Safranin

Paper towel

Forceps

Method:

Using a permanent marker, write the unknown organism number and your name on the edge of the microscope slide

Transfer the organism with an inoculating loop to the slide, add a drop of water mix and let it air dry. Heat fix right after that

Place the microscope slide on a boiling water bath that is equipped with a staining rack. Cut a piece of paper towel the same size as the microscope slide and cover the smear

Add malachite green staining solution in a moderate amount. Heat it over the water bath until the stain steams (which is about 5-6 minutes). Add more malachite green solution as soon as it starts evaporating. Do not let the slide dry out

Using the forceps, remove the paper towel and allow the slide to cool down. Rinse the slide for 30 seconds with water

Counterstain the slide with safranin for 60-90 seconds

Rinse for 30 seconds with water. Blot dry with paper towel

Examine under microscope and use immersion oil if necessary

Results:

No results recorded for this staining procedure

Discussion and conclusion:

Seeing as gram positive cocci of Streptococcus, Staphylococcus and Micrococcus are non-spore forming it was unnecessary for me to do the staining procedure. In normal results free spores and endospores stain green; vegetative cells will stain red under the microscope.

Capsule staining

Principle:

When bacteria have a slimy layer surrounding them it is usually referred to as a capsule. The thickness and composition varies from specie to specie. Organisms with a thick capsule are usually more pathogenic than organisms with no capsule. They cannot be stained with simple stains. Two staining procedures have proved to be effective namely: Anthony's and Graham & Evans procedure. Anthony's procedure involves a primary stain such as crystal violet giving the bacterial cell along with its capsule a dark purple colour. The capsule is non-ionic and the primary stain is unable to adhere. Copper sulphate is used as a decolourizer and at the same time as a counter stain. It is absorbed into the capsule and will give it a light blue or pink appearance. No heat-fixing is necessary as this is likely to shrink the capsule. An e.g. of a capsule stain:

Materials:

Organism being isolated on TSA media

Crystal violet

20% copper sulphate solution

Microscope

Immersion oil

Microscope slides

Permanent marker

Paper towel

Inoculating loop

Method: (Anthony's method)

Label in the corner of the microscope slide your name and organism number with a permanent marker

Transfer a loop full of culture aseptically to a microscope slide using an inoculating loop. Air dry the slide

Place the microscope slide on a staining rack and flood the slide with crystal violet. Let it stand for about 4-7 minutes

Using the 20% copper sulphate solution, rinse the slide properly

Blot dry with paper towel

Examine under immersion oil (capsules will appear as faint halos around dark cells)

Results:

No results recorded for this procedure

Discussion and conclusion:

Due to the fact that there was no copper sulphate solution I wasn't able to do my test, thus I will have to make use of the biochemical testing to make a final determination of which organism I have.

Flagella staining

Principle:

Bacteria use threadlike organelles called flagella for locomotion. They have a slender appearance and can thus only be examined under an electron microscope. To view them with a light microscope the flagella thickness should be increased using a mordant such as tannic acid or potassium alum and staining them with basic fuchsin, parasosaniline, silver nitrate or crystal violet. Flagella staining is of great importance for identification of bacteria. There are different types of flagella found on an organism, these are examples:

Materials:

18 hour tryptic soy agar slants of unknown organism

Permanent marker

Inoculating loop

Acid cleaned glass slides with frosted ends

Distilled water

Microscope

Immersion oil

Boiling water bath

Bunsen burner

Pasteu pipettes with pipettor

Wast stain - solution A + B

Difco's spot test flagella stain

Method:

Mark the corner of a microscope slide with the organism number and your name with a permanent marker

Transfer with an inoculating loop the bacterium from a turbid liquid at the bottom of the slant aseptically to the centre of the microscope slide and mixing it with 3 drops of distilled water. Spread the suspension gently over a 3cm area, using the inoculating loop

Let the slide air dry for 15 minutes

Cover the smear with solution A for 4 minutes

Rinse properly with distilled water

Place a paper towel over the microscope slide and soak the slide with solution B. For 5 minutes heat the slide in a boiling water bath in an exhaust hood with the fan on

Remove the paper towel and rinse off with distilled water and thereafter flooding the slide with distilled water and letting it stand for 1 minute

Rinse once more with distilled water and carefully shake the excess water off

Let the slide air dry

Examine the slide in an oil immersion objective. The best specimens will be seen at the edge of the smear

Results:

No results recorded for this procedure

Discussion and conclusion:

Staphylococcus, Micrococcus and Streptococcus are all non-motile and thus explain why no flagella would be found on the slide so there was no need for me to do the procedure.

Discussion of the staining properties and morphology of the isolate

Up to this point I know that I have gram positive cocci that occur in short chains, pairs and clusters. This would give me an indication that it is Micrococcus, Staphylococcus or Streptococcus. They are all non spore-forming and non-motile which means no flagella. They are also acid-fast negative. Due to the fact that Micrococcus turns the medium of tryptic soy agar to a red colour where my colonies were white and my medium colour stayed the same, I can assume that it is not Micrococcus and can thus eliminate this possibility.

Biochemical tests used for the identification

Catalase test

Principle:

Bacteria that contain flavoproteins that reduce O2 will produce hydrogen peroxide. They are toxic due to their oxidizing agents and destroy cellular components rapidly. A lot of bacteria have enzymes that protect themselves against toxic O2 products. If bubbles are present in the catalase test it represents a positive test and if no bubbles occur it is negative for the catalase test. An e.g. of a catalase test showing the positive and negative reactions for this test.

Materials:

18-24 hour tryptic soy cultures of unknown organisms

Tryptic soy agar slants (TSA)

3% hydrogen peroxide (H2O2)

Bunsen burner

Inoculating loop

Pasteur pipettes with pipettor

Incubator at the 35

Test tube rack

Permanent marker

Microscope slides

Method:

Label the TSA slants with your name, organism number and date

Aseptically heavily inoculate the bacterium into the test tube with a streak inoculation

Incubate for 18-24 hours

Remove growth of the slant aseptically with an inoculating loop and place it on the slide. Mix with a drop of H2O2

Examine for bubbling

Results:

Bubble formation occurs in moderate amount

Discussion and conclusion:

Due to the bubble formation I can assume that my organism is catalase positive. That means I can basically rule out the possibility that my organism might be a Streptococcus, but I will do more tests to show which specie of Staphylococcus I have.

Coagulase test

Principle:

Coagulases are able to clot blood plasma. It is not required for pathogenicity. Coagulase producing organisms form a fibrin clot around themselves and protect themselves by avoiding attack from the host's defence system using this technique. Citrate and EDTA are used to act as anticoagulants and preventing false positive results. If they are unclottes after 4 hours they are coagulase negative. This is an example of a coagulase test indicating the positive and negative results. The positive results are on the left side and the negative results are on the right hand side:

Materials:

Tryptic slants of unknown organism

Citrated rabbit plasma

Water bath at 35

Inoculating loop

Bunsen burner

Pipette 1ml

Permanent marker

Incubator at 35

Test tube rack

Method:

Aseptically, heavily streak inoculate the tryptic slants with the unknown organism

Incubate for 18 - 24 hours in an inverted position

Label a microscope slide with your organism number and your name

Aseptically transfer from the tryptic slants your organism to the labelled microscope slide

Take the rabbit plasma from the water bath and add 2 drops with the pipette to the microscope slide. Mix thoroughly

Examine the slide almost immediately

Results:

By adding the rabbit plasma my mixture on the microscopes slide turned slightly cloudy and it has thickened.

Discussion and conclusion:

From my results that I have gathered I can assume that my organism is coagulase positive. This means that I have a Staphylococcus aureus organism due to the fact that Staphylococcus epidermidis is coagulase negative. I will do one more test to finally conclude which organism I have.

Mannitol Salt Agar test

Principle:

This medium is both selective and differential and is mainly used for the isolation of Staphylococcus pathogens. The 7,5 % NaCl is a high salt concentration medium and prevents other mixed cultures from growing. Mannitol is there to show if the organism is fermentative. When they are fermentative they will produce acid turning the medium yellow indicating a mannitol positive reaction. Non-fermentative organisms will be mannitol negative. This procedure is most helpful to differentiate between Staphylococcus aureus and Staphylococcus epidermidis. The following is an e.g. of Staphylococcus aureus which is fermentative and will grow on the mannitol salt agar.

Materials:

Unknown organism

Petri plates

Mannitol salt agar

Distilled water

Weighing boat

Spatula

Weighing scale

200 ml glass bottle

Inoculating loop

Autoclave

Water bath at 52

Incubator at 35

Bunsen burner

Autoclave protective gloves

Permanent marker

Method:

Weigh off enough mannitol salt agar to prepare the required amount of petri plates and mix it with with the required amount of distilled water in the glass bottle

Autoclave the mixture for 15 minutes to sterilize the media

When the pressure of the autoclave has dropeed to 0 the glass bottle can be transferred to the boiling water bath using the protective gloves to do so and allowing the media to cool down in the water bath

When the medium has cooled down it can be poured aseptically to the petri plates. Let it stand for about 20 minutes and wait for it to solidify

After it has solidified, label the petri plates in the corner with your name, date and organism number

Streak inoculate the agar using the Bunsen burner, inoculating loop and unknown organism and working aseptically

Place the petri plates in the incubator in an inverted position for 24-48 hours

Examine for results

Results:

Growth has occurred on mannitol salt agar changing the agar to a yellow colour as well as the colonies formed on the agar.

Discussion and conclusion:

Due to the fact that my organism has grown on mannitol salt agar I can be sure that I have a Staphylococcus specie. Furthermore, due to the fact that my agar changed to a yellow colour it means that my organism is fermentative producing an acid, thus I can establish that my organism is definitely Staphylococcus aureus.

Susceptibility test

Antibiograms

Principle:

Susceptibility testing for bacterial pathogens may be organized into a summary table or antibiogram used mainly by clinicians, pharmacists, infection control personnel and microbiologists. Antibiograms can be used to raise awareness of resistant problems, supporting optimal empiric therapy, and reducing inappropriate antibiotic usage.

Using the Kirby-Bauer method (sensitivity disk method) one can determine antibiotic susceptibility. This method uses antibiotics that are impregnated onto paper disks and which are placed on a Mueller-Hinton agar plate by making use of forceps. The plates are then incubated for 16-18hours and the zone of inhibition around the disk is measured. Small zones of inhibition or no zones indicate that the pathogen is resistant against the strain of antibiotic. Factors such as size of inoculums, distribution of inoculums, incubation time, depth of agar, diffusion rate of antibiotics, concentration of antibiotics and growth rate of bacterium's should be controlled for ultimate success. The method can also test sensitivity to antimicrobial agents and synthetic chemotherapeutics. The disks include the following antibiotics: 1) Penicillin G (10 units - Pink), 2) Cefoxitin (30µg - White), 3) Eryhromycin (15µg - Red), 4) Cotrimoxazole (25µg - White), 5) Ampicillin (10µg - Grey), 6) Chloramphenicol (30µg - Green), 7) Gentamicin (10µg - Salmon), and 8) Cefuroxime (30µg - Primrose).

Materials:

Mueller-Hinton agar plates and antibiotic disks

Sterile swabs

Tryptic soy broth cultures of unknown organism

35 water bath

Forceps

Metric ruler

Permanent marker

Bunsen burner

Method:

Label the lids of the Mueller-Hinton agar plates with your name, date and organism number

Using a sterile cotton swab take up the unknown organism and streak it on the Mueller-Hinton agar plate 3 times and rotate the plate 60 after each streak. Then run the swab around the edges of the agar ensuring that all the surfaces are covered. Allow the plate to dry for about 5-10 minutes

Insert the antibiotic disks on the paper sheath by using sterile forceps. Press the antibiotic disks gently on the culture to ensure contact using sterile forceps. Avoid pressing the disk into the agar or moving the disk around on the agar plate

Incubate for 16-18 hours at 35. These plates must not be inverted when incubated.

Measure the zones to the nearest mm to determine the inhibition of the organism

Results:

Disk 1: 18mm inhibition diameter

Disk 2: 20 mm inhibition diameter

Disk 3: slight growth occurred

Disk 4: growth occurred

Disk 5: 17mm inhibition diameter

Disk 6: 20mm inhibition diameter

Disk 7: 26mm inhibition diameter (biggest inhibition)

Disk 8: growth occurred

Discussion and conclusion:

According to my results Erthromycin, Cotrimoxazole and Cefuroxime will have no effect in inhibiting Staphylococcus aureas. What will inhibit Staphylococcus aureus though, would be Penicillin G, Cefoxitin, Ampicillin, Chloramphenicol and Gentamicin. Gentamicin will most probably have the best effect in inhibiting the organism due to its big inhibition zone, thus Gentamicin should be used for the inhibition of Staphylococcus aureus.

Refrences:

Principles of Microbiology M&S by Methrotra and Sumbali pg 108

Microbiology Laboratory excercises (5th ed J.P Harley & L.M Prescott).pdf-Adobe Reader pg43, 52, 57-59, 63-65, 67-70, 93-95, 99-102, 156-160, 169-170, 173-174.

Microbiology I - Practical Manual (Compiled by Prof LE Anelich - 1995) pg21, 67-70, 77.

http://www.amrita.vlab.co.in/?sub=3&brch=73&cim=208&cnt=1

http://www.faculty.mc3.edu/year/ML/ml-10.htm

http://www.amrita.vlab.co.in/?sub=3&brch=73&sim=720&cnt=1

http://www.ijpmonline.org/article.asp?issn=0377-4929;year=2012;volume=55;issue=3;spage=361;epage=364;aulast=Thool

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