(Autonomous) - observation of microorganisms commonly found in food: Gram ... from the manufacture of fruit juices, ... fermentation of fruit sugars to alcohol

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    Department of Microbiology

    St Aloysius College, Mangalore-575003.


    Re -accredited by NAAC A Grade CGPA3.62

    College with Potential for Excellence






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    1. Microscopic observation of microorganisms commonly found in food: Gram smear

    preparation and Tease mount preparation of fungi.

    2. Observation and identification of bacterial types and fungi.

    3. Preparation of culture media used in food examination.

    4. Microbial examinations of foods: Isolation of bacteria and Fungi from different food


    5. Tests for milk: Dye Reduction tests-Methylene blue reduction and Resazurin test.

    6. Tests for milk: Lactose and Lactic acid estimation in milk.

    7. Direct Microscopic count of bacteria.

    8. Water examination: Multiple tubes Method. Water examination: PA method.

    9. Detection of adulterants in Food: Tests for milk adulterants and Ghee.

    10. Detection of adulterants: Tests for honey and jaggery, sweetmeats and ice cream.

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    Materials Required:

    1. Clean glass slides

    2. Inoculating loop

    3. Bunsen burner

    4. Bibulous paper

    5. Microscope

    6. Lens paper and lens cleaner

    7. Immersion oil

    8. Distilled water

    9. 18 to 24 hour cultures of organisms


    1. Primary Stain - Crystal Violet

    2. Mordant - Grams Iodine

    3. Decolourizer - Ethyl Alcohol

    4. Secondary Stain - Safranin


    Part 1: Preparation of the glass microscopic slide

    Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from

    the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides

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    with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until

    ready for use.

    Part 2: Labeling of the slides

    Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to

    clearly designate the area in which you will prepare the smear. You may also label the slide

    with the initials of the name of the organism on the edge of the slide. Care should be taken that

    the label should not be in contact with the staining reagents.

    Part 3: Preparation of the smear

    Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture

    on the slide. Spread by means of circular motion of the inoculating loop to about one

    centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A

    satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.

    Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline

    solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a

    bacterial colony and gently stir into the drop of water/saline on the slide to create an emulsion.

    Swab Samples: Roll the swab over the cleaned surface of a glass slide.

    Please note: It is very important to prevent preparing thick, dense smears which contain an

    excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass

    through, thus making it difficult to visualize the morphology of single cells. Smears typically

    require only a small amount of bacterial culture. An effective smear appears as a thin whitish

    layer or film after heat-fixing.

    Part 4: Heat Fixing

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    Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the

    sample to more readily take up stains.

    Allow the smear to air dry.

    After the smear has air-dried, hold the slide at one end and pass the entire slide through the

    flame of a Bunsen burner two to three times with the smear-side up.

    Now the smear is ready to be stained.

    Please Note: Take care to prevent overheating the slide because proteins in the specimen can

    coagulate causing cellular morphology to appear distorted.

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    Part 5: Gram Stain Procedure

    1. Place slide with heat fixed smear on staining tray.

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    2. Gently flood smear with crystal violet and let stand for 1 minute.

    3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

    4. Gently flood the smear with Grams iodine and let stand for 1 minute.

    5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The

    smear will appear as a purple circle on the slide.

    6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop

    by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-


    7. Immediately rinse with water.

    8. Gently flood with safranin to counter-stain and let stand for 45 seconds.

    9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

    10. Blot dry the slide with bibulous paper.

    11. View the smear using a light-microscope under oil-immersion.

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    The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of

    staining and observing fungi and is simple to prepare. The preparation has three components:

    1. Phenol: kills any live organisms;

    2. Lactic acid : It preserves fungal structures, and

    3. Cotton blue : It stains the chitin in the fungal cell walls.

    Lactophenol Cotton Blue Solution is a mounting medium and staining agent used in the

    preparation of slides for microscopic examination of fungi. Fungal elements are stained


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    Glass slide




    Lactophenol Cotton blue

    Preparation of lactophenol cotton blue (LPCB) slide mounts

    1. Place a drop of seventy percent alcohol on a clean microscope slide.

    2. Material from cultures of filamentous fungi should be removed using a stiff inoculating wire

    not the loop used for manipulations with bacteria or yeasts.

    3. Flame the wire by holding it upright in the hottest part of the Bunsen flame, just above the

    blue cone, until the whole length of the wire glows red hot.

    4. You must ensure that the inoculating wire has cooled before placing it in a fungal culture it

    should have cooled sufficiently after approximately ten seconds.

    5. Remove the cap from the tube but do not put it on the bench. Kill any contaminating

    microorganisms by flaming the neck of the tube.

    6. Remove a small amount of the culture. For fungal cultures, it is often useful to take a little of

    the agar medium together with the fungus. In any case, the material should be disturbed as

    little as possible when being transferred to the slide.

    7. Flame the neck of the tube once more and replace the cap.

    8. Immerse the fungal material in the drop of seventy percent alcohol. This drives out the air

    trapped between the hyphae.

    9. Tease out the material very gently with mounted needles.

    10. Do not forget to sterilise the inoculating wire and the needles after use by heating to red

    heat in a Bunsen flame

    11. Fungal structures are readily visualised after staining with a lactophenol cotton blue dye


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    12. Before the alcohol dries out add one or at most two drops of the stain. A common fault is to

    add too much to the preparation. Holding the coverslip between your index finger and

    thumb, touch one edge of the drop of stain with the edge of the coverslip.

    13. Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is

    now ready for examination.

    14. Make the initial examination using a low power objective lens. The thinner parts of the

    preparation, generally around the edges of the mounted material, will yield the best images.

    15. Switch to a higher power 40X objective for more detailed examination of spores and other





    Learning Objective

    Identify a fungal unknown based on colonial morphology and microscopic appearance.


    In this experiment, you will be provided with a number-coded pure culture of a representative

    fungal organism for cultivation and subsequent identification.



    Number-coded, 7-day Sabouraud broth spore suspensions of Aspergillus, Mucor, Penicillium,

    Alternaria, Rhizopus, Cladosporium, Fusarium, and Yeast.


    One Sabouraud agar plate per student.


    Lactophenolcotton-blue solution.


    Bunsen burner, dissecting microscope, hand lens,sterile cotton swabs, glass slides, coverslips,


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    loop, and glasswa