Objectives

Introduction

It is especially important to work with a well characterized strain if the microorganism is used for food preparation, as well as in antibiotic production where the product is to be taken internally. For the same reason that a student will not lick an incubated plate that was exposed to the air, the presence of a large number of contaminants may present a potential health hazard, especially when the exact natures of the contaminants are unknown. Licking that agar plate probably will not kill the student; however, the probability of fatality certainly increases with higher numbers of organisms. Conversely, the probability of a wasted fermentation run due to contamination is directly proportional to the number of microorganisms in the environment, which in turn is directly related to the cleanliness of the laboratory.

The need for a clean working environment in biochemical engineering studies must be stressed again, for cleanliness is the prerequisite to any meaningful work. (This obsession is not the result of the instructor having had bad toilet training as Sigmund Freud may have concluded.) As demonstrated by the plate transfer process, many parts of the aseptic procedures require occasional exposure to the surrounding environment. Since our laboratory cannot be made totally sterile economically, it is imperative that the room be kept clean. The use of a laminar hood, which creates an air curtain to reduce the chance of contaminants drifting into the working space enclosed by the hood, is highly recommended if it is available. However, a laminar hood itself must still be maintained in a clean condition.

List of Reagents and Instruments

A. Equipment

• Erlenmeyer flasks
• Culture tubes
• Petri dishes, 100*15mm
• Bunsen burner
• Inoculation loop
• Autoclave
• Balance
• Magnetic Stirrer or stirring rod
• Incubator, 37ºC
• Thermometer
• Refrigerator

B. Reagents

• Nutrient media:
  • Yeast extract
  • Peptone
  • Agar
  • Glucose
  • Lactose
  • Sucrose
  • Dipotassium phosphate
  • Eosin
  • Methylene blue
• HCl, 0.1 N solution
• KOH, 0.1 N solution

Procedures

1. Petri dish preparation:
   • Mix the following nutrient ingredients in proportion to the amount required. Note that a Petri dish pour requires about 12 ml and a slant needs 6 ml. Adjust the pH to the desired value (pH=5.0) with 0.1N HCl or 0.1N KOH. If a large number (more than 5) of plates are to be poured, mix the nutrient in 1-2 liter flasks. It is difficult to handle flasks larger than 2 liters with one hand. If only a few (less than 5) are needed, the nutrient can be divided and poured into test tubes, each holding enough media for one Petri dish. The advantage of using test tubes is that they can be autoclaved separately and may later be heated to melt the agar in a beaker of boiling water. The liquefied agar may then be poured into a Petri dish directly from the test tube. However, the use of test tubes is not practical when making large quantities of agar plates.

         YPG (east extract- Peptone- Glucose) Agar
         ---------------------------
         Yeast extract         5g
         Peptone              10g
         Glucose               5g
         Agar                 15g
         ---------------------------
         Add water to make 1 liter
     

         LB (Luria-Bertani) Agar
         ---------------------------
         Yeast extract         5g
         Tryptone             10g
         NaCl                 10g
         Agar                 15g
         ---------------------------
         Add water to make 1 liter
         If necessary, adjust pH to 7.5 with NaOH.
     

     For ampicllin plates, add enough 25g/L stock solution of ampicillin aseptically to obtain a final concentration of ~50mg/L. One can pipet 0.05ml of ampicillin stock solution to each plate containing 25mL of agar just before agar solidifies. Because ampicillin plates stay fresh for only a few weeks, alternatively, one can pipet 0.05mL of ampicillin stock solution to a solidified agar plate, smear it evenly with a sterile L-shaped glass rod, and let the plate stand for 10 minutes to absorb ampicillin. The latter method has the advantage of freshness. Ampicillin is heat-labile; so, sterilize the ampicillin stock solution with a filter and store at -20°C.

         EMB (Eosin- Methylene Blue) Agar
         -------------------------------
         Peptone                10g
         Lactose *               5g
         Sucrose *               5g
         Dipotassium phosphate   2g
         Agar                   13.5g
         Eosin                   0.4g
         Methylene blue          0.065g
         -------------------------------
         Add water to make 1 liter
         *5 g/l of glucose may be substituted for lactose and sucrose.
     

   • Cover the flask with a beaker or a piece of aluminum foil, and cover the culture tubes with caps, cotton plugs, or gauge plugs. Autoclave the media for 20 minutes. The heat of sterilization will dissolve the agar. Place a watch glass over the flask.
   • Set the media on a bench top and cool until the flask can be handled with your bare hands. Wrap paper towel around the neck of the flask when pouring agar into Petri dishes just in case the media reheats the glassware as the hot liquid passes through the neck in the process of pouring. Of course, one cannot pour out a block of solidified agar if the flask is cooled excessively. The agar will solidify at approximately 42ºC; thus, the temperature window for handling the liquefied agar is quite narrow. If the agar starts to solidify at the bottom as a result of not pouring fast enough, the flask can be gently heated with a flame to raise the temperature and melt the agar.
   • Stack sterile Petri dishes in a row, three or four per stack, on a disinfected lab bench. The number os dishes per stack can be adjusted according to the size of the student's hand. With the right hand holding the neck of the flask, open the cover of the lowest Petri dish with the left hand just wide enough to pour the media. (The top cover is the larger dish of the two; the actual dish is the smaller one.) Pour about 12 ml into each Petri dish. If too little agar is poured, there may not be enough to cover the dish or the agar plate will dry up easily. If too much is poured, the cover dish will come in contact with the nutrient agar, leaving no room for microbial growth. The plates are rendered useless either way. After pouring the bottom dish, pour the second one from the bottom, and so on until the entire stack is poured. Proceed to the next stack until all dishes are poured.
   • Immediately clean the flask with hot water before the residual agar solidifies, or have fun painstakingly scraping out the solidified agar from the flask.
   • Leave the plates undisturbed until the agar solidifies. The plates may now be streaked.
2. Slant tube preparation:
   • Mix and prepare the YPG nutrient agar as in the preparation of Petri dishes. Pour 6 ml of the nutrient into a test tube. Plug the test tube with a cotton ball or a cap. Culture tubes with screw caps may also be used.
   • After autoclaving the media for 20 minutes, the tubes are placed in a slanted position to allow the agar to solidify. These tubes are called slants.
3. Plate streaking with inoculum from a liquid suspension: This entire sequence must be repeatedly practiced with an empty Petri dish and plain water until it can be carried out smoothly without referring back to this write-up.
   • Gently shake the flask or tap the bottom of the culture tube with your finger to suspend microbial cells in the broth that are to be transferred. Watch out for the broth level and do not wet the cotton plug when shaking.
   • Light a Bunsen burner and flame an inoculation loop until the wire turns red hot. This will kill all the bugs on the loop surface. Slightly flame the handle as well to minimize the chance of contamination.
   • Cool the loop for about 5 seconds.
   • While the loop is cooling, with the thumb and index finger of the right hand holding the inoculation loop and with the left hand holding the flask or culture tube, take out and hold onto the plug with the baby finger and palm of the right hand. Quickly pass both the neck and rim of the flask through the flame.
   • Carefully dip the loop into the broth that contains the microorganism to be streaked. Note that only the small loop at the tip needs be dipped, not the entire extension of the wire leading to the loop and definitely not the handle. Since the handle is not really well sterilized, do not touch the side of the flask or culture tube with the handle.
   • Flame the neck and rim of the flask or tube as before. After lightly flaming the bottom and side of the plug, place the plug back into the mouth of the flask. Note that the side and bottom of the plug never comes in contact with anything but air; the plug is never placed on a table.
   • Put down the flask and pick up a Petri dish with the left hand. Flip open the cover plate with the middle finger and the thumb. Gently stroke 3-5 parallel lines at one corner of the plate with the loop to spread the cells. (Lines marked A in the following Figure.) Re-close the Petri dish; the best results are achieved when the dish is recovered immediately following the streaking. Do not gouge the agar with the loop; a light touch is all that is necessary to deposit the culture onto the plate.
   • Flame the loop as before and cool it for about 5 seconds. Gently touch the free surface of the agar plate to cool it further to avoid killing all the organisms upon the initial contact. Spread the cells further with 5-6 strokes (Lines B in the Figure); start each stroke by crossing the original set of lines.
   • Repeat the above step twice more to create Lines C and D, flaming the loop each time before streaking.
   • When done streaking, flame the loop for the last time before returning it to the bench. This is to prevent the spread of microorganisms on the bench. This last step is especially crucial when working with pathogenic strains.
   • Incubate the inoculated plate upside-down at 37ºC for 48 hours. Store the plate, also upside-down, in a refrigerator thereafter.
4. Plate streaking with inoculum from another plate: Repeat the above steps of plate streaking, except that the inoculum is taken from another Petri dish.
   • Find a single colony on the original agar plate that is located nearby Line D and is physically isolated from all other colonies on the same plate. This colony is homogeneous in the sense that all the cells degenerate from the same parent. Thus, this can be considered as a pure culture.
   • Flame the loop and lift a minute amount of culture from the original plate. Remember that one need not see the colony of organisms on his loop to prove that that the microorganisms are indeed there. There is already too much if one can see the culture on the inoculation loop.
   • Transfer the cells in the loop to a new agar plate by following the same set of steps involved in streaking a plate as before. Incubate the inoculated plate.

Discussions

Uninoculated agar plates may stacked and stored upside down in a refrigerator for about 3 months if not used immediately. The inverted position ensures that water does not condense on the bottom of the cover plate and retards the loss of moisture. An inoculated agar plate is also incubated and stored upside down to retain moisture. In addition, the water condensate can act as a medium for transport of cells from one location on an agar plate to another. The slants may be stored in a refrigerator for over a year if culture tubes with screw caps are used.

Incubation of the inoculated plates should last for about 48 hours. They are stored in a refrigerator, and the culture can stay viable for over one month in agar plates or for one year in sealed slants. Prolonged storage of the plates in the incubator dries up the agar and kills the culture. The severe loss of moisture can be visually detected by the appearance of macroscopic cracks that develop in the agar plate.

The used agar plates and other biological wastes should be autoclaved in a pan to destroy all biological activities before being disposed.

Questions

1. Why is the agar in test tubes allowed to solidify in a slanted position?
2. During the preparation of an streak plate, why flame the loop between different sets of strokes?