MICROBES

TITLE: TRANSFER & ISOLATION TECHNIQUES OF MICROBIOLOGY

OBJECTIVE:

1.    To transfer bacteria aseptically between tubes of growth media

2.    Aseptically perform a streak plate resulting in isolated colonies.

MATERIAL:

Mixed culture (Broth, agar slant, agar plate)

Bunsen Burner

Inoculating Loop

Sterile broth, agar slant & agar plate

Alcohol 70 %

PROCEDURE:

Inoculation of a broth culture (broth to broth transfer)

1.    The sterile nutrient broth is labeled with the source of culture and initials.

2.    The loop is sterilized.

3.    Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.

4.    The loop is inserted into sterile broth tube and gently swirl. The loop is sterilized.

5.    The broth is incubate at 37’C for 24-48 hours.

6.    The broth is observed for turbidity. The result is recorded in table at the end of procedure section.

Inoculating on agar slant (Broth to slant transfer)

1.    The sterile nutrient agar slant is labeled with source of culture and initials

2.    The loop is sterilized.

3.    Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.

4.    The loop is inserted into the sterile agar slant tube and starting at the base of the slant, the loop is draw up the slant. The agar not penetrate. The loop is sterilize.

5.    The slant is incubated at 37’C for 24-48 hours.

6.    The slant for growth is observed. The result is recorded in the table at end of the procedure section.

Streak plate ( Plant culture to Plant transfer)

1.    The sterile nutrient agar slant is labeled with source of culture and initials

2.    The loop is sterilize.

3.    Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed

4.    The agar plate is lifted from the lid and streak about the half of plate. The loop is  be in parallel to the agar surface to prevent digging into gouging the agar.

5.    The plate of lid is returned. The loop is sterilized. The agar plate is lifted and make one streak into the inoculated portion of the plate. By streaking, the remaining one-fourth of the uninoculated plated is finished.

6.    The plate of lid is returned. The loop is sterilized. The agar plate is lifted and make one streak into the second inoculated portion of the plate. By streaking, the remaining one-fourth of the uninoculated plated is finished. The loop is sterilize.

7.    The plate is placed in a 37’C incubator for 24-48 hours. The growth is observed and result is recorded in table provided at the end of the procedure section.

Inoculating on agar slant (slant to slant transfer)

1.    The sterile nutrient agar slant is labeled with source of culture and initials

2.    The loop is sterilized.

3.    Appropriate aseptic technique is used, the loopful of broth from the mixed culture tube is removed.

4.    The loop is inserted into the sterile agar slant tube and starting at the base of the slant, the loop is draw up the slant. The agar not penetrate. The loop is sterilize.

5.    The slant is incubated at 37’C for 24-48 hours.

6.    The slant for growth is observed. The result is recorded in the table at end of the procedure section.

DISCUSSION:        

A mixed culture contains two or more bacterial species that are known and can be easily separated based on cultural or biochemical characteristics. Culturing techniques provide a means for maintaining adequate nutrition for the organisms so they can continue to survive. As organisms grow in a culture they consume theavailable nutrients and periodically need to be transferred to fresh media to continue to grow. Certain culturing techniques not only provide the organisms with a fresh supply of nutrients but also allow for the separation of bacterial cells to obtain isolated colonies. These culturing procedures are known as isolation techniques.

Streaking is a technique used in microbiology to isolate a pure strain from a single species of microorganism, often bacteria. A microbiological culture can be grown so that the organism can be identified, studied, or tested. A sterile cotton swab or inoculation loop is sterilized and dipped in a broth or patient specimen containing many species of bacteria.

There is some precaution we must take attention, in the streaking procedure, a sterile loop or swab is used to obtain a microbial culture. The inoculating instrument is then streaked lightly over an agar surface. On the initial section of the streak, many microorganisms are deposited resulting in confluent growth, which is growth over the entire surface of the streaked area. However, because the loop is sterilized between streaking different sections, or zones, fewer and fewer microorganisms are deposited as the streaking progresses. Finally, only an occasional microorganism is deposited, because the streaking process dilutes out the sample that was placed in the initial section.

            To obtain good results with this technique, the agar surface should be smooth, moist, and free of contamination. However, excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media, can collect on the inside of the lid and sides of the plate. If this water drops down onto the agar surface, spreading and merging of colonies can occur. This is why one should invert the plates after streaking them and when they are incubated.

                       

CONCLUSION:

Based on the result above, student can transfer bacteria aseptically between tubes of growth media and aseptically perform a streak plate resulting in isolated colonies. Thus, the objective achieved.

QUESTION:

1.    How can you tell growth has occurred in a broth culture?

It becomes turbid, the odor may change and if there is an indicator (that is a pH indicator) the color may change.

2.    What is the purpose of agar?

Agar is used to solidify microbiological media. Having a solid media allows microbiologists to see colonies of bacteria this eases the identification of a single microbe in a mixed population it also allows the selection of variants in a population, it perhaps allows identification from colonial morphology and enumeration of bacteria. Solid media in lawn cultures (spread plates) are used to calculate zones of diffusion/inhibition. Agar is used rather than other substances as relatively few microbes can manufacture the digestive enzymes required to break it down, so the solid media does not become liquid.

3.    At what temperature does agar liquefy and solidify?

0.7% agar will solidify at around 30 deg C but it will stay solid after that at much higher temperatures and liquefy at low than 30’C.

4.    Why is liquefied agar cooled to 60’ C before adding organisms?

To improve the motility of medium agar

5.    What is primary purpose of the streak plate?

Isolate a single cell by dilution

REFFERENCES:

www.who.int/csr/resources/publications/​drugresist/IIIAMRManual.pdf

www.epa.gov/epawaste/hazard/testmethods/​sw846/pdfs/9131.pdf

fda.gov/Food/ScienceResearch/​LaboratoryMethods/.../ucm073598.htm

PARASITOLOGY 1

TITLE: MICROSCOPIC EXAMINATIONS

INTRODUCTION:

Microscopy is the technical field of using microscopes to view samples or objects which cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). The microscopic portion of the ova and parasite examination can be determined by microscopic examination. These can be divided into three distinct steps which the direct mount, the concentration and the permanent stain smear.

PRINCIPLE:

The direct wet mount is performed to detect motile protozoan trophozoites and to determine cellular morphology.

OBJECTIVE:

To describe commonly used methods for microscopic examination for parasites.

MATERIALS:

Sample material, clean glass microscope slide, 22 x 22 mm coverslip, 0.85% NaCl solution,

Lugol’s iodine solution, Eosin and Biohazard discard container.

METHOD:

·         Saline Mount.
1. With an applicator stick, a small amount (-2mg) of fecal sample is transferred and is emulsified in the saline drop.
2. A 22 x 22 mm cover slip is placed over the suspension.
3. By using the lower objective (10x), the entire surface area of the cover slip for parasites.
4. The high dry objective (40x), is used for investigation of suspicious objects.

·         Iodine Mount
1. An iodine stained mount is prepared in the above manner, one drop of Lugol’s iodine for the saline is substituted.
2. A drop of iodine is also added to the edge of cover slip of a previously examined saline mount. The iodine is diffused into the stool-saline mixture, killed the organisms and stained the cellular elements.

·         Eosin Mount
1. An Eosin stained mount is prepared in the above manner, one drop of Eosin for the saline is substituted.

QUALITY CONTROL:

Known the positive specimen should be processed to verify the recovery organisms at least quarterly and the results should be properly recorded.

EXPECTED RESULT:

Motile protozoan trophozoites will demonstrate their characteristics motility in saline wet mounts. Addition of iodine solution and eosin kill the organisms and stain the celluar elements for easier recognition

RESULT AND OBSERVATION:

STAINS	TROPHOZOITE	CYST
NORMAL SALINE	Endoplasma stains blue green.	Chromatoidal bar are readily seen, cyst wall smooth and thin, glycogen vacoule cannot be seen.
IODINE MOUNT	Iodine kills the microorganism.	Yellowish or light brown, nucleus and karyosome prominently stains dark brown, chromatoidal bars did not stain, glycogen vacoule stains dark brown.
EOSIN MOUNT	Eosin kills the microorganism	Background material appears red

DISCUSSION:

            Based on the experiment conducted, the direct examination of a clinical specimen is the most rapid, cost-effective diagnostic aid in the laboratory. Microscopic examination of a sample is used to identify the motility and morphology of microorganisms, fungal elements, protozoan trophozoites, and helminth eggs and larvae. The visual examination assists the laboratory and the clinician in quickly identifying the causative agent of infection so treatment may begin. Proper sample collection is important in diagnosing infection.

            The expected result and the result obtained is same. In normal saline,the cyst shown  that chromatoidal bar are readily seen (if any), cyst wall smooth and thin, and glycogen vacoule cannot be seen the cyst. For trophozoite, endoplasma stains blue green. In addition, the Iodine shown the cyst yellowish or light brown, nucleus and karyosome prominently stains dark brown, chromatoidal bars did not stain and glycogen vacoule stains dark brown. The trophozoites it will kills the microorganism. In Eosin, the cyst will appears background material appears red and trophozoites will kill the microorganism.

This shown the result is accurate. To obtained the good result, some precaution we have to take care of when transfer the small amount of the fecal sample. The volume of saline also affected the result. We have to make sure the volume of saline is not too many. When placing the coverslip, be sure that there no air bubble entered the covered place. In other hand, when observing the specimen in the microscope make sure it is overcome within the direction up and down.

            If the precaution have been taken, the false positive results and the false negative result will occur. For the false positive result, the problem that related is maybe the specimen is preserve for the long time. This can make the result occurs positive while the actual result is negative. Moreover, when observing in microscope we see and think that it is the cyst or the trophozoites but it is not the truly ones. For the false negative result, the problem outcomes is lack or human skills and the substances or chemical involve in the experiment is expiry and cannot been used.Besides that, too much of saline can make the specimen become replenished and the microbes cannot be seen.

            Based on the discussion above state, the principle of this experiment is performed  to detect motile protozoan trophozoites and the cellular component morphology is determined. Last but not least, the objective is to describe commonly used methods for microscopic examination for parasites is achieved.

 CONCLUSION:

The commonly used methods for microscopic examination for parasites is described.

REFERENCES:

http://www.vin.com/proceedings/Proceedings.plx?CID=WSAVA2007&Category=&PID=18212&O=Generic

http://en.wikipedia.org/wiki/Microscopic_examination

http://www.ok.gov/health/documents/2010%20PROC%20-%20G%20Wet%20Prep%20updated%2001-10.pdf

QUESTION:

1.    Do not use 100x lens objectives during observation of direct wet preparation slides. Why?

The cyst and trophozoites can be seen within 10x lens objectives. These can be seen the full of morphology of cyst and trophozoites. If  100x lens use, the structure of the cyst and trophozoites will appears bigger and hard to identify it.

2.    List all of the organelles movement for protozoa and give ONE (1) example of species respectively.

Cilia, pseudopodia and flagella. The cytoplasm of certain cells, such as phagocytes, or of certain unicellular organisms, especially amoebas, that serves in locomotion and phagocytosis. The species is amoeba.

3.    Give TWO (2) general safety rules applied for this experiment.

Careful when using electric microscope.

Avoid contaminated chemical during the experiment.

 TITLE: CONCENTRATION TECHNIQUE

1.    ZINC SULFATE FLOTATION TECHNIQUE

INTRODUCTION:

The diagnosis of intestinal parasitic infection is confirmed by the recovery of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. The ability to detect and identify intestinal parasites in fresh stool specimen depends on immediate collection, transportation, and examination by the laboratory; often, these time requirements from passage of the specimen to examination cannot be guaranteed. Substituted zinc sulfate for the mercuric chloride in the preparation of it's Z-PVA fixative. Although mercuric chloride based PVA provides the best fixation, tests with Z-PVA have shown it to be a viable alternative, and far more effective than copper sulfate based PVA.

PRINCIPLE:

The flotation procedure facilitities the separation of protozoantrophozoites, cyst and certain helminth eggs background debris through the use of high specific gravity zinc sulfate solution.

OBJECTIVES:

To determine the purpose of each concentration techniques

To recognize commonly used method for concentration technique.

To recognize methods used for less commonly encountered parasites.

MATERIALS:

Preserved fecal material, 15 ml conical centrifuge tube, funnel/gauze filter device, 0.85% saline solution, centrifuge, 1.18 or 1.20 specific gravity zinc sulfate solution, clean microscope slide, 22 X 22 mm coverslip, biohazard discard container.

METHODS:

1.    A pea size sample of fresh stool (about 4g) into 10 ml of 5 or 10% formalin is transfers. The specimen is mixed thoroughly and let stand for a minimum of 30 minutes for fixation. The mixture is resuspanded if the specimen is already preserved.

2.    A sufficient quality of the specimen is strained through wet gauze into a 15 ml conical centrifuge tube.

3.    The remainder at the tube with 0.85% saline is filled and centrifuge for 10 minute at 500Xg. The amount of sediment obtained should be approximately 0.5 to 1.0 ml.

4.    The supernantant fluid is decant and the sedimentation is resuspended and centrifuge in 0.85% saline for 10 minute at 500Xg. This can be eliminated if the supernatant is resulting from step 3 is light tan to clear.

5.    Decant the supernatant and the sediment is resuspended into 2 ml of zinc sulfate. The tube with additional zinc sulphate within 2-3mm of the rim is filled.

6.    The specimen is centrifuge for 20 minute at 500Xg. The complete stop of centrifuge is needed without interference. The result shown is in the two layer.

7.    Without removing the tube from centrifuge, the drop of the surface film with a Pasteur pipette is withdrawl by using wire loop.

8.    The 22 x 22mm coverslip is added to the slides. The iodine is added to enhance the morphology details.

9.    The lower objectives (10x) is used, the entire area of the coverslip is systemically scan.

10.  The suspicious is observed, and can be made with (40x) lens objectives.

QUALITY CONTROL:

The specific gravity of the zinc sulfate should be frequently monitored. When fresh stool is received for processing, a specific gravity of 1.18 should be maintained. If formalin preserved is used, the specific gravity of the zinc sulfate solution should be adjusted to 1.20

Concentrate known positive specimens and verify organism recovery at least quarterly and after the recalibration of the centrifuge. All QC results must be properly recorded.

EXPECTED RESULT:

Protozoan trophozoites and or cyst and some helminth eggs and larvae may be recovered. Operculated eggs and heavy helminth eggs will not float on the zinc sulfate. These organism may be found in the sediment. Oocyst of C.parvum may also be seen in the concentrate sediment.

RESULT AND OBSERVATION:

Protozoan trophozoites and or cyst and some helminth eggs and larvae have been recovered.

DISCUSSION:

           

            Fecal flotation methods levitate the diagnostic products of endoparasitic organisms (eggs, larvae, oocysts, and cysts) in the feces of animals by use of suspension medium with a higher specific gravity than the parasite products. Parasite eggs, cysts, and oocysts are concentrated on the surface of the medium because of their lighter density. The result is a clean preparation for microscopic examination with a minimal amount of distracting fecal debris.

            Zinc Sulfate solution (sp gravity 1.18) is used preferentially to identify potential infections with parasitic protozoan species like Giardia lamblia and for the recovery of delicate larval stages of lungworm parasites like Oslerus and Filaroides in dogs, and Aleurostrongylus in cats. Studies in our laboratory demonstrated that Zinc Sulfate as a flotation medium for detecting Giardia infections is nearly 2 times more reliable than Sheather’s Sucrose.

            Centrifugation has been advocated as an important aid to flotation recovery of diagnostic parasite structures for microscopic examination. The Companion Animal Parasite Council recommends centrifugal flotation as a “Best Practice” for processing all fecal samples in veterinary medical practice. Despite the scientific evidence supporting its use, reluctance to employ the centrifugal flotation technique in routine parasitologic examinations persists.

There are some precaution that we must to take attention while doing this experiment. To obtained the good result, some precaution we have to take care of when transfer the small amount of the fecal sample. The volume of saline and zinc sulfate also affected the result. We have to make sure the volume of salineand the zinc sulfate is not too many. When placing the coverslip, be sure that there no air bubble entered the covered place. In other hand, when observing the specimen in the microscope make sure it is overcome within the direction up and down. When using the centrifuge make sure it in the specific gravity to avoid the sample become hemolysis.

            If the precaution have been taken, the false positive results and the false negative result will occur. For the false positive result, the problem that related is maybe the specimen is preserve for the long time. This can make the result occurs positive while the actual result is negative. Moreover, when observing in microscope we see and think that it is the cyst or the trophozoites but it is not the truly ones. For the false negative result, the problem outcomes is lack or human skills and the substances or chemical involve in the experiment is expiry and cannot been used. Besides that, too much of saline can make the specimen become replenished and the microbes cannot be seen.

CONCLUSION:

Based on the result obtained, we can determine the purpose of each concentration techniques. Besides that, recognize commonly used method for concentration technique. Moreover, we can  recognize methods used for less commonly encountered parasites.

REFERENCES:

http://www.vet.utk.edu/diagnostic/parasitology/Detections%20of%20Parasitic%20Infections%20by%20Fecal%20Exam

http://society.kisti.re.kr/~kspa/kjp/abstracts/1964_52.html

http://veterinarynews.dvm360.com/dvm/article/articleDetail.jsp?id=59865&&pageID=2

QUESTION:

1.    List all the advantages and disadvantages for each of the concentration technique.

Zinc Sulfate Flotation Technique

            Advantages

            -effectively eliminating the majority of contaminating debris

            Disadvantages

-unreliable for the recovery of heavier, operculated eggs of some trematodes, unfertilized Acardis eggs and nematode larvae.

Formalin-ethyl Acetate Sedimation

Advantages

-easy to perform and inexpensive

Disadvantages

-most flotation techniques are that the walls of eggs and cysts will often collapse, thus hindering identification.

Sheather’s sugar flotation technique

Advantages

-effective for recovery of Cryptosporidium and Isospora cyst

Disadvantages

-most flotation techniques are that the walls of eggs and cysts will often collapse, thus hindering identification.

Explain the purpose of respective reagent used in concentration technique as stated below.

i)              Formalin

- Specimens preserved in formalin can be tested directly (wet mount, immunoassay, chromotrope stain, UV fluorescence) or can be concentrated prior to further testing

ii)             Ethyl acetate

- Used when the feces contain a large amount of fat (which make use of a flotation method difficult) or when you are specifically looking for trematode or cestode eggs.

iii)            1.18 or 1.20 specific gravity zinc sulfate solution

- higher specific gravity than the organisms to be floated so that the organisms rise to the top and the debris sinks to the bottom.

TITLE: PERMANENT STAIN

            2. WHEATLY’ MODIFICATION OF GOMORI’S TRICHOME STAIN

INTRODUCTION:

It is generally recognized that stained fecal films are the single most productive

means of stool examination for intestinal protozoa. The permanent stained smear

facilitates detection and identification of cysts and trophozoites and provides a

permanent record of the protozoa found. The trichrome technique of Wheatley

for fecal specimens is a modification of Gomori’s original staining procedure for

tissue.

PRINCIPLE:
The stain is rapid, simple procedure that produces uniformly well-stained smears of intestinal protozoa, as well as human cells, yeast cells and artifact material. It facilitates the identification of trophozoites and cysts, and the confirmation of species, and can be a permanent record of organisms recovered.

OBJECTIVE:

·         To identify trophozoites and cyst that has been stained through appropriate staining procedure.

·         To determine the purpose of permanent stains.

·         To determine the most suitable permanent stains for each species of protozoa.

MATERIALS / APPARATUS:.
Prepared fecal slides, 10 coplin jars, 70% ethanol plus iodine, 70% ethanol, trichrome stain, 90% glacial acetic acid alcohol ,95% alcohol, absolute alcohol, Carboxylene, Xylene, Permount, Microscope slides, 22 x 40 mm coverslips and c ompound microscope.

PROCEDURE:
1. Prepared slide is placed into the first coplin jar. Stain smears according to the staining time given:

COPLIN JAR REAGENTS STAINING TIME
1. 70% ethanol plus iodine 10-20 minutes
2. 70% ethanol 5 minutes
3. 70% ethanol 5 minutes
4. trichrome stain 8 minutes
5. 90% glacial acetic acid alcohol 3-5 seconds (briefly dip in & out)
6. 95% ethanol Rinse briefly
7. 95% ethanol Rinse briefly
8. Absolute alcohol 5 minutes
9. Carboxylene 5 minutes
10. Xylene 10 minutes

2. The slide is remove from coplin jar 10 and several drops of permount is added onto smear. A 22 x 40 mm coverslip is placed carefully on the smear. Avoid bubble formation.
3. Smear is allowed to dry overnight or 1 hour at 37ºC.
4. The smear microscopically is examined with 100X objective. At least 200 to 300 oil immersion fields are examining.

QUALITY CONTROL:

·         Prepare and stain a smear of PVA fixed fecal specimen containing protozoa or PVA-preserved negative stool specimen to which buffy coat cells have added weekly.

·         Include a QC smear when the decoloring reagent has been changed a new reagent have been added weekly.

·         Cover all staining dishes to prevent evaporation.

·         If xylene becomes cloudy, replace before staining.

EXPECTED RESULT:

·         Protozoan trophozoites and cyst will be readily seen.

·         Cytoplasm of trophozoites or cyst stain blue-green. Chromatin material, chromatoidal bodies, red blood cells (ingested) and bacteria stain red or purplish-red.

·         Background material appears green; larvae or egg stain red.

RESULT AND OBSERVATION:

DISCUSSION:

Based on the result state, the result is accurate but there are some limitation when using this techniques. The permanent stained smear is not recommended for staining helminth eggs or larvae; they are often too dark (excess stain retention) or distorted. However, occasionally they are recognized and identified. The wet smear preparation from the concentrate is the recommended approach for identification of helminth eggs and larvae.

The smear should be examined with the oil immersion lens (100X) for the identification of protozoa, human cells, Charcot-Leyden crystals, yeast cells, and artifact material. These cells and other structures are normally quantitated from the examination of the permanent stained smear, not the wet-smear preparations (direct wet smear or concentration wet smear) (rare, few, moderate, many, packed). With the exception of Blastocystis hominis, intestinal protozoa are not quantitated on the report. This high-magnification (oil immersion, total magnification, 1000X) examination is recommended for protozoa, particularly for confirming species identification.

With low magnification (10X objective), one might see eggs or larvae; however, this is not recommended for the permanent stained smear as a routine approach.In addition to helminth eggs and larvae, I. belli oocysts are best seen in wet preparations (concentration wet smears prepared from formalin-preserved, not PVA-preserved, material).Cryptosporidium spp. oocysts will generally not be recognized on a trichrome stained smear (modified acid-fast stains or the immunoassay reagent kits are recommended).All solutions need to be changed periodically, to prevent carry-over and/or watering down of the solutions. Carry-over of the solutions may cause lack of contrast and/or cloudiness on the slide.

If the Trichrome Stain weakens, it may be strengthened by exposing it to air overnight, or by replenishing with fresh stain.When using the Modified Trichrome Blue Stain, data has indicated that centrifugation at 500xG for 10 minutes dramatically increases the number of microsporidial spores available for staining from concentrate sediment.The result show the Cytoplasm of trophozoites or cyst stain blue-green. Chromatin material, chromatoidal bodies, red blood cells (ingested) and bacteria stain red or purplish-red.In addition, background material appears green; larvae or egg stain red.

The false positive results and the false negative result will occur. For the false positive result, the problem that related is maybe the specimen is preserve for the long time. This can make the result occurs positive while the actual result is negative. Moreover, when observing in microscope we see and think that it is the cyst or the trophozoites but it is not the truly ones. For the false negative result, the problem outcomes is lack or human skills and the substances or chemical involve in the experiment is expiry and cannot been used. Besides that, too much of alcohol can make the specimen become replenished and the microbes cannot be seen.

CONCLUSION:

Result show the Cytoplasm of trophozoites or cyst stain blue-green. Chromatin material, chromatoidal bodies, red blood cells (ingested) and bacteria stain red or purplish-red.

In addition, background material appears green; larvae or egg stain red. The trophozoites and cyst that has been stained through appropriate staining procedure was indentified. Moreover, the purpose of permanent stains is can be known. Besides that, the most suitable permanent stains for each species of protozoa is determined.

REFERENCES:

www.med-chem.com/procedures/WheatleyTRICHROME.pdf

insidesurgery.com/medical.../gomoriwheatley-stain-trichrome-stain/

www.hardydiagnostics.com/catalog2/.../Parasitology%20Mini-Cat.pdf

           

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