Several aspects of general microbiology are presented here to “limber up” the fingers and the mind before encountering exercises involving diagnostic bacteriology.  APPLYING GOOD LABORATORY TECHNIQUE IS SOMETHING THAT CANNOT BE STRESSED ENOUGH.  It is strongly urged that the following sections be read,  reread, understood, and kept available for ready reference.
 
 

 GENERAL LABORATORY SAFETY INSTRUCTIONS

 In this laboratory course, you will be handling pathogens.  Treat them with respect and caution at all times. The safety techniques you learned in general microbiology are exactly what you will need to apply in this course.  A few  safety measures  extreme importance are listed below.

 1.  The practice of personal cleanliness is important in any laboratory.  It is a safe practice to wash your hands frequently and especially before leaving the laboratory.  Use plenty of soap and hot water.  At times, it is advisable to bathe your hands in a 2% disinfectant before washing with soap and water.

 2.  Eating  and drinking in the laboratory are strictly forbidden as a precaution against accidental infection.  Never place fingers, pencils, labels, or any other objects or materials in your mouth for the same reason.

 3.  Your working area must be free of extraneous articles, e.g., books, purses, etc.  With the disinfectant solution provided, wipe the table top work area before and after you work.

 4.  Wearing a plastic apron or laboratory cotton coat is mandatory while working in the laboratory.  Shoes are also required.

 5.  If infectious material is spilled on the table top or floor, flood the entire contaminated area and splash area immediately with disinfectant and allow to stand for 10 minutes or longer before mopping.  Notify the instructor or preparation room personnel at once of accident.  Instructions for decontamination of clothes and shoes will be given if needed.  Caution nearby workers to avoid the contaminated area until it is properly cleaned.

 6.  If contaminated tubes or glass containers are broken, cover a wide area with 2% disinfectant.  Let stand for 30 minutes, then collect with a dust pan, and autoclave the broken glassware before discarding in the broken glass bin.

 7.  In the preparation of smears for stains, place slides in the lid or the bottom of a petri plate.

 8.  Use safety pipette device at all times when transferring microbes or any other liquids with pipettes.

 9.  Remove all cultures, etc. from the incubator immediately after proper incubation periods.  Remove all cultures or infectious materials from the storage facility as soon as they are no longer needed.  Place all discard cultures and other contaminated materials to be discarded in the designated and marked area so that they may be autoclaved before they are washed.

 10. All materials such as plates, tubes etc placed in incubators or some storage facility must be labeled with your  initials, ID #, date, and content material.
 

TEST TUBE MANIPULATIONS

 1.  The test tube should be held in one hand and the inoculating loop in the other.

 2.  The cap is removed with the little finger of the hand holding the loop handle.  The cap should remain removed from the top of the tube only for the time needed to manipulate within the tube.  In other word make sure the cap is on the tube at all times except when working in the tube.

In a tube to tube transfer, each tube is handled individually.

First, the tube containing the inoculum is handled as described above.  The cap  is removed with the little finger of the hand holding the inoculating loop.  After charging the loop, the cap is put back on the tube and the tube is positioned   back into the test tube rack.  The second tube is then picked up and the transfer is made with the charged loop.
 

SMEAR PREPARATION

 Place slide for smear preparation into a lid or bottom of an empty plastic petri dish.  Smears of bacteria for staining are most satisfactory when a bit of growth from an agar plate or slant culture is emulsified to light turbidity in a drop of tap water on a grease free slide, then spread.  If a broth culture is to be stained, 2-3 loopfuls of the culture is removed after mixing to suspend the cells evenly and spread on the grease-free slide.  To prepare a smear from an agar culture follow the following instructions.

 1.  Place a small drop of water on a clean, grease-free slide.  With a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water until a very small amount of turbidity is noted in the drop.  Flame the loop to remove the excess growth from the inoculating loop.  Then again using the loop, suspend the bit of growth in the drop and spread it evenly over a portion of the slide to make a thin film with light visible turbidity or cloudiness.

 2.  Allow the film to air-dry, then “heat fix” it by passing the bottom of the slide through the burner flame for a second or two, until the slide is just uncomfortably warm (but not burning hot) when the heated portion is touched to your hand.

GRAM STAIN

 The gram stain is one of the most useful differential stains in bacteriology, including diagnostic medical bacteriology.  The differential staining effect is believed to depend upon certain fundamental differences in the physical structure and the chemical composition of bacterial cell walls.  The cultures to be stained should be young  (12 - 18 hours) and grown on a sugar-free medium (e.g., nutrient agar).

   Solution I:    2% crystal violet and 0.8% ammonium oxalate
   Solution II:   2% aqueous solution of iodine
   Solution III:  95% ethanol (7 parts) and acetone (3 parts)
   Solution IV:  2.5% safranine

  Procedure

  1.  Cover the smear with crystal violet (Solution I).  Allow the mixture to stain for 2 minutes; then tilt the slide to drain excess stain.

  2.  Cover the film with iodine (Solution II).  Allow to act 2 minutes; then wash with tap water.

  3.  Destain with acetone-alcohol (Solution III) by tilting the slide and dripping the solution onto the slide until the washings are practically colorless (about 10 seconds). Wash well with tap water.

  4.  Counterstain the smear with safranine (Solution IV) for 1 minute; then wash and blot dry.  Examine under oil immersion with maximum light in order to distinguish colors.

NOTE:  Gram-positive cells stain purple and gram-negative cells stain pink or red.  Reliable results may be expected only with young or actively-growing cultures.

NOTE: Try to standardize the times that each reagent is applies to the smear.  This will allow the final gram stain color to be fairly constant. Also be very careful with the destaining agent (acetone-alcohol).  You can over destain and you can under destain.  Be sure that the destaining reagent gets in contact with all of the smear.  If you have a properly made smear that is very faint, it shouldn’t take more than about 10 seconds of destaining contact. If your smear is thick, more time will be necessary.  Just because you have stopped dripping the destaining reagent does not stop the destaining process. The water wash is used to stop the destaining process.

NOTE:  Many gram-positive bacteria (Bacillus, Staphylococcus, Streptococcus, etc.) have the tendency to rapidly lose the ability to retain the dye complex after exponential growth ceases.  Thus, the physiological state of cells play an important role.
 

USE OF THE MICROSCOPE
 

 1. Moving and transporting the microscope. The microscope is stored in a special cabinet. Each microscope has its own cubicle with very little extra room. Be careful when removing or replacing the microscope in that the eyepieces could get damaged at the top of the storage cubicle.  The microscope always should be removed and transported from its housing or storage place by grasping the arm of the microscope with one hand and supporting the base with the other. It should never be sharply tilted since the eyepieces are not fixed in the body tube and may drop to the floor. Always set the microscope down gently to avoid damage.

 2. Cleaning. The exterior lenses of the eyepieces and the objectives should be superficially cleaned after using the microscope. Use only microscope lens paper to wipe the lenses. If you suspect that interior lenses are dirty, consult your instructor. Do not attempt to clean them yourself.   Routinely, the microscope lenses need to be cleaned thoroughly, especially the objective lenses.  This can be accomplished using cotton swabs, commercial lens cleaner, and canned air.

 3. Positioning the slide. Place the slide on the stage with the specimen (smear or coverslip preparation) over the middle of the hole in the stage. Be certain that a stained smear is turned up toward the objective (much time may be wasted looking at the wrong side of a slide). If a smear is thin, faint, or small, encircle it with a marker on the underside of the slide to aid in centering the smear. Secure the slide on the mechanical stage with the “clip” device.

 4. Illumination. Work with the oil immersion lens generally requires a powerful source of illumination which usually is supplied by a halogen lamp. Switch on the lamp by actuating the main switch on front left side of base. The lamp voltage can be adjusted between 2 V and 6 V by turning knurled ring in front right side of base, which has a relative designation range of 1 to 9. This potentiometer for brightness is provided with a catch position of 4.8 V at a relative scale reading of 6. The intensity achieved there will be sufficient for the majority of examinations carried out and ensures a long lamp life. If higher light intensities should be required, the range above the catch position (red scale mark) can be used.

 5. Focusing. Four rules must be learned and never forgotten when using the microscope:
    (a) only use the coarse adjustment focusing knob with the scanning and low power objective;
    (b) never focus with the coarse adjustment when using the high dry or oil immersion lenses;
    (c) never allow an objective lens to jam into or even to touch the slide or coverslip;
    (d) never force the adjustments.

NOTE: If a smear is faint, place a mark with a marker or wax pencil near the smear to aid in finding the plane of focus.  First focus on the mark, then move to the smear area.

    (a) Focusing with SCANNING and LOW POWER  lenses.  (3.2X and 10X objectives) The               wide-field lens below condenser must be in the light path for these objectives. While looking from the side, use the coarse adjustment to raise the stage until it stops. Then look through the eyepieces; slowly focus    downward with the coarse adjustment until the specimen comes into view.  Then bring into sharp focus with the fine adjustment and adjust the light intensity to obtain maximum detail in the image. Less light is required for low power magnification than for higher magnification.

    (b) Focusing with HIGH DRY lens. (40X) Remove wide-field lens from optical path. Since the microscope is parfocal, rotate the revolving nosepiece until the high power objective snaps into place and increase the amount of light, and bring the specimen into sharp focus by slight rotation of the fine adjustment knob in one direction or the other, as required.

    (c) Focusing with the OIL IMMERSION lens.(100X objective) To observe the specimen with the oil immersion objective, revolve the high power objective slightly to the side so that a drop of immersion oil may be placed on the specimen. Then revolve the oil immersion objective until it snaps into place. The objective should be in the oil but must not touch the slide. Increase the light intensity as required and rotate the fine adjustment knob to obtain a sharp focus of the specimen. If necessary, make further adjustments to obtain optimum illumination.

 NOTE: In most cases, the preparation needs to be observed only under oil immersion. It is recommended that the specimen be located and centered in the field with the low power objective (10X). Then oil is added, and the oil immersion objective is rotated into position. Clarity of the specimen is obtained through proper adjustment of lighting and the fine adjustment.

 NOTE: Once the slide has oil on its surface and has been observed under the oil immersion lens, be careful not to get oil on the high dry lens by switching to the high dry lens.  The high dry and oil immersion lenses are about the length and therefore will dip into the oil if oil is present in the viewing area.  If you do get oil on the high dry lens be sure to clean it thoroughly with lens cleaner, etc.

 6. Mechanical difficulties. Certain mechanical difficulties, real or apparent, often are encountered by the student in operating the microscope. The student should consult the instructor for aid if (1) either the coarse adjustment and/or the fine adjustment become difficult to turn; (2) the objective appears to drop slowly out of focus while the specimen is being examined, or the stage perceptibly drops after being racked up with the coarse adjustment.

 NOTE:  A more common problem is the failure of the fine adjustment to turn in the direction required for sharp focusing. This indicates that it has been screwed to the limits of its threads, either upward or downward, as the case may be. Screw it back to about one-half the thread distance, use the coarse adjustment to raise or lower the objective sufficiently to bring the specimen into view, then refocus with the fine adjustment.
 

STREAKING A PLATE
 

Follow the attached protocol at the end of handout:

NOTE:  It is not necessary to divide the plate into three sectors with the lines, numbers, or asterisks, etc.  You can imagine where the sectors are on the plate and the other designations. The main aspect is to follow the  major instructions of flaming the loop between each sector, keeping the  streaks as close together as possible and do not press the loop against the agar surface.  When streaking hold the inoculating loop handle between only your index finger and your thumb.  No other fingers should be involved in holding the loop handle. Additionally,  hold the inoculating loop handle at the far back end.   If you do not explicitly follow these later instructions you will press too hard and not get the proper isolation of colonies.
 

PIPETTING

To use the pipetting device-

1.  Be sure the plunger is depressed.

2.  Remove a pipette from the pipette can.

NOTE:  The pipette should only be touched or handled with one hand at the upper end or mouth end of the pipette.  Never  handle the pipette with two hands.  Touching the tip end with your hand  will contaminate the pipette, whether it is the sterility or chemical contamination aspect.  Once a pipette is removed from the can, never put it back.

3.  Insert the mouth end of the pipette into the collar of the pipette pump. Push it gently past the stabilizing fingers at the end of the pump’s collar. A gentle twisting motion will ease insertion of the pipette. Push the pipette in until it is well seated in the soft chuck.

NOTE:  Do not force or put excess pressure to seat the pipette, due to the fact that  pipettes break easily and can cause serious cuts.

4.  Place pipette tip into the depth of the liquid to be pipetted.

5.  Rotate wheel counter clockwise, the liquid will rise in the pipette.

6.  Stop when the required amount has been exceeded.

7.  Be sure to raise the tip of the pipette out of the liquid

8.  Turn knurled wheel in a counterclockwise direction to lower the level of the iquid in the pipette and stop it at  the proper mark or volume.

9.  To transfer the volume of liquid,  place the tip end of the pipette right above the liquid into which it is being transferred. Do not place the tip into the liquid.

10.  For fast release of the liquid, press the side lever until the pipette is empty.

       (a)  To release the liquid in small amounts or at a slow flow, turn the knurled wheel slowly in the     counter clockwise direction.
 
 

CULTURE MEDIA

 All organisms require food for maintenance and growth.  Bacteria are no exception.  The bacteriologist must supply the necessary nutrients to bacteria if they are removed from their natural environment.  Culture media are employed for the isolation and maintenance of pure cultures and are also used for identification of bacteria according to their biochemical and physiological properties.  In general, the culture medium must supply suitable carbon and nitrogen sources, minerals, and in some instances, small quantities of certain “growth factors.”

 There are two major types of culture media based on nutritional supply, complex and chemically-defined (“synthetic”).   The purpose of a complex medium is to supply an excess of all nutrients required for growth of each viable cell with minimum lag and maximum growth rate, i.e., a medium of high productivity.  Body fluids, tissue infusions and extracts, peptones, etc, are frequently included in complex media, as they usually contain all nutrients and growth factors, known and unknown, which may be required for maximum growth of an organism.  Chemically-defined culture media are of known composition, qualitatively and quantitatively.  They are compounded of inorganic and, often, of organic compounds, the formulae of which are known.

 Some of the most clever and inventive media belong to the categories of selective and differential media.  These media are designed for special microbial groups and they have extensive applications in isolation and identification.  They can permit in many cases and in a single step, the preliminary identification of a genus or even a species.

 A selective medium contains one or more agents that inhibits the growth of a certain microbe or microbes (A, B, C) but not others (D), and thereby encourages or selects microbe D and allows it to grow.  Selective media are very important in primary isolation of a specific type of microorganism from samples containing a highly mixed population - for example, feces, saliva, skin, water, and soil.  They hasten isolation by suppressing the background organisms and favoring growth of the desired ones.

 A differential medium can grow several types of microorganisms, but it is designed to highlight differences among these microorganisms.  Differentiation shows up as variations in colony size and color, in media color changes, and in the parities in appearance are due to the type of agents added and the way the cells react to them.. For example, when microbe X metabolizes a certain substance not used by organism Y, then X will cause a visible change in the medium and Y will not.  The simplest differential media demarcate two reaction types - showing the use or nonuse of a particular nutrient, or one type organism (colony) reacting with a dye or some specific component while the other one not.

The different types of culture media, their ingredients, and their purposes are of utmost importance in diagnostic bacteriology.  This type information will be exemplified in a number of the laboratory exercises, some informative examples are given below to demonstrate the principle of differential and/or selective media.
 

Eosin Methylene Blue Agar (EMB)  contains various nutrients (e.g., peptone) to  support the growth of a number of bacteria .  However, other ingredients (at specific concentrations) are included to make it selective and differential.  It is designed to inhibit gram-positive bacteria with methylene blue and also to differentiate between gram-negative lactose and nonlactose fermenters.  Lactose fermenters develop colonies that are colored due to the absorption of the eosin-methylene blue complex which  forms in the presence of acid.  Nonlactose fermenter are colorless.  Escherichia coli (a member of the mixed acid fermentation group)  colonies that  demonstrate a green metallic sheen.  Enterobacterand Klebsiella (members of the butylene glycol group) colonies are generally mucoid and pink due to the fact that their cells possess capsules and belong to the butylene glycol fermentation group and thus produce less organic acids from the lactose in the medium, thus absorb    less complex.

Mannitol Salts Agar contains only mannitol as a fermentable carbohydrate, the pH indicator phenol red for detection, and a high concentration of salt to inhibit undesired organisms.  It has been observed that coagulase positive staphylococci grew luxuriantly, producing colonies with yellow zones.  Nonpathogenic staphylococci produce small colonies with no color change of the surrounding medium.  Other bacteria are generally inhibited, making possible the use of a heavy inoculum without danger of overgrowth.

Blood Agar is used for the isolation and cultivation of many pathogenic    bacteria such as Staphylococcus and Streptococcus.  Microorganisms can be differentiated as to their “hemolytic” type on this medium, based on observed changes in the blood agar surrounding each organism’s colonies.  Blood agar is normally an opaque rich red color.  Beta-hemolytic organisms will produce a zone of clearing around their colonies, due to an actual lysis of red cell membranes and cell destruction by bacterial exoenzymes known as hemolysins.  Alpha-hemolytic organisms will produce a zone of green, olive, or brown discoloration around their colonies, due to oxidative effects of peroxide wastes on heme (pigmented portion of hemoglobin).  Gamma-hemolytic organisms will exhibit no detectable change in the blood agar around their colonies.
 
 

                                                 E. coli                               B. subtilis                          St. aureus
 

Cellular morphology
     and gram stain
 
 
 
 
 

Colonial morphology
 

         Blood Agar
 
 
 
 
 

         Mannitol Salts
 
 
 
 
 
 

         Nutrient Agar
 
 
 
 
 
 

         EMB Agar