Practical Manual Food Microbiology

EXPERIMENT 3 CULTIVATION AND SUBCULTURING OF MICROBES

Structure
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 Objectives Introduction Principle Involved Preparation and sterilization of nutrient medium
3.3.1 3.3.2 3.4.1 3.4.2 3.5.1 3.5.2 Method Procedure Materials Required Procedure Materials Required Procedure

Preparation of slants, stabs & pouring of Petri plates with nutrient agar Sub culturing of cultures Observations Results Precautions

3.0

OBJECTIVES

After attending to this experiment, we shall be able to: describe different types of culture media; prepare nutrient medium; prepare agar plate, agar slant and agar stab; perform aseptic technique; and do sub-culturing in liquid and solid media.

3.1

INTRODUCTION

In natural environment, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. In working with microorganisms, we must also have a sterile nutrient containing-medium in which to grow the organisms. Anything in or on which we grow a microorganism is termed a medium. A sterile medium is one which is free of all life forms. It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed.

3.2

PRINCIPLE INVOLVED

Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying to isolate or identify. Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have adapted to the habitats most suitable for their needs, in the laboratory, however, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. Depending on the type and combination of nutrients, different categories of media can be made.

3.2.1 Categories
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Complex media are rich in nutrients, they contain water soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone) but since the exact composition is unknown, the medium is called complex. Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known. Selective/differential media are media based on either of the two categories above supplemented with growth-promoting or growth-inhibiting additives. The additives may be species- or organism-selective (e.g., containing a specific substrate for growth of desired micro-organism or an inhibitor for perceptive growth of other species).
Table 3.1: Types of media and their use Media Complex Defined Selective Differential Enrichment Purpose Grow most heterotrophic organisms Grow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays Suppress unwanted microbes, or encourage desired microbes Distinguish colonies of specific microbes from others Similar to selective media but designed to increase the numbers of desired micro-organisms to a detectable level without stimulating the rest of the bacterial population For promotion of growth of obligate anaerobes

Introduction to the Basic Microbiology Laboratory Practices

Reducing

The mixture of necessary nutrients can be used as a liquid medium known as broth or a solidifying agent can be added. "Agar agar" is a natural polysaccharide produced by marine algae and is the most commonly used solidifying agent added to media (end concentration usually 1.5 % w/v). This medium is known as agar. Finally, in working with micro-organisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique. When we use solid media i.e. agar for culturing the microorganisms, we can prepare slants , stabs in screw cap tubes or test tubes, we can also use glass petri plates for making pour plate, spread plate or streak plate. The agar plates are used for maintaining pure culture for sub culturing purposes. These are prepared by pouring out the agar into the plate and then allowed to solidify whereas slants and stabs are prepared by pouring media in test tubes while in liquefied state, which are then allowed to harden in slanting position( for stabs they are left as such). Agar slants provide more surface area for microorganisms. Stabs and slants are easier to store and transport than Petri dishes. Moreover the slant has a slope which is easier to streak than a horizontal surface. Agar stabs or agar deep tubes are used for the study of gaseous requirements of microorganisms. Agar plates are prepared by pouring the melter (100oC) and cooled (45oC) liquefied media into sterile petri dishes that provide large surface area. These are used for isolation, counting and study of microorganisms. Agar plates after solidification are to be kept in an inverted position to prevent dropping of water vapour on hardened agar plate.
Table 3.2: Composition of nutrient medium Component Quantity

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Practical Manual Food Microbiology

Beef extract Peptone NaCl2 Distilled water Agar pH= 7.0 + 0.2

(g/l) 3 5 3 1000ml 15g (to be added for nutrient agar)

3.3 PREPARATION AND STERILIZATION OF NUTRIENT MEDIUM

II. 3.3.1 Material Required
Reagents: Beef extract (lab lemco) ,peptone, NaCl2, distilled water, agar Equipment and glassware: Pipettes with pipette aids, Pipette and petri dish containers, autoclave, sterilized conical flasks, test tubes, screw cap tubes

3.3.2 Procedure
1. Weigh the ingredients and dissolve in 800ml of distilled water, by gentle heating. 2. Cool the contents and adjust the pH to the desired level. 3. Filter the contents through non-absorbent cotton or muslin cloth and adjust the volume to 1000ml. 4. Dispense into sterilized tubes and flasks. Plug them tightly with nonabsorbent cotton and autoclave at 15psi for 15-20 minutes.

3.4 PREPARATION OF SLANTS, STABS AND POURING OF PETRIPLATES

3.4.1 Material Required
Reagents: Sterile Nutrient agar Equipment and glassware: Petri dishes/plates- glass or plastic (at least 15 x 90 mm), screw cap tubes, cotton plugs, laminar air flow chamber, autoclave

3.4.2 Procedure
1. All the glassware was sterilized by oven drying method i.e. 160oC for 2hrs. 2. 2-3ml of nutrient agar was taken in screw cap test tubes in non-sterile form. 3. These test tubes were autoclaved at 121oC for 15min. 4. For slant preparation few test tubes were inclined and allowed to solidify. 5. For stab preparation the test tubes containing sterilized agar were allowed to stand in an upright position and agar was allowed to solidify at the base. 6. For pour plate preparation, after the addition of dilute sample, 15-18 ml of molten agar was added to petri plate. The contents were mixed by rotating in clockwise and anticlockwise direction. The petri plates were then allowed to cool for solidification of agar. In a similar fashion, plates can be prepared for streaking and spread plate but there is no addition of sample prior to pouring agar. The sample is loaded on the surface of Petri plate either with glass rod bent at 90o or an inoculating needle. All the plates i.e. pour, spread, or streak are then incubated in inverted position.

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3.5

SUB-CULTURING OF CULTURES

III. 3.5.1 Material Required

Cultures: 18-24 hour old nutrient agar slant or nutrient broth cultures of E.coli Reagents: Sterile nutrient agar plates, Sterile nutrient broth tubes (10ml). sterile agar slants and stabs Equipment and glassware: Inoculating loop and needles, Bunsen burner or Laminar air flow chamber

Introduction to the Basic Microbiology Laboratory Practices

IV. 3.5.2 Procedure

I. Sterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum. II. Remove the inoculum. a. Removing inoculum from a broth culture 1. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil. 2. Remove the cap of the pure culture tube with the little finger of your loop hand. 3. Very briefly flame the lip of the culture tube. This creates a convection current which forces air out of the tube and preventing airborne contaminants from entering the tube. The heat of the gas burner also causes the air around your work area to rise, and this also reduces the chance of airborne micro-organisms contaminating your cultures. 4. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculum. 5. Again flame the lip of the culture tube. 6. Replace the cap. b. Removing inoculum from a plate culture 1. Sterilize the inoculating loop in the flame of a gas burner. 2. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. In case of slant or stab, remove the cap near the flame. 3. Scrape off a small amount of the microbial growth organisms and close the lid or cap. III. Transferring the Inoculum to the Sterile Medium. a. Transferring the inoculum into a broth tube: 1. Pick up the sterile broth tube and remove the cap with the little finger of your loop hand. 2. Briefly flame the lip of the broth tube. 3. Place the loopful of inoculum into the broth, and withdraw the loop. Do not lay the loop down. 4. Again flame the lip of the tube. 17

Practical Manual Food Microbiology

5. Replace the cap. 6. Re-sterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again.

Fig. 3.1: Inoculation of broth tube

b. Transferring the inoculum into a petri plate: 1. Lift the edge of the lid just enough to insert the loop. 2. Streak the loop across the surface of the agar medium using the pattern shown in fig. 3.2 (a). These streaking patterns allow you to obtain single isolated bacterial colonies originating from a single bacterium or arrangement of bacteria. 3. In order to avoid digging into the agar as you streak the loop over the top of the agar you must keep the loop parallel to the agar surface. Always start streaking at the "12:00 position" of the plate and streak side-to-side as you pull the loop toward you. 4. After each sector, the loop is sterilized (flamed) and cooled. Rotate the plate counter clock-wise, so that you are always working in the "12:00 position" of the plate. This keeps the inoculating loop parallel with the agar surface and helps prevent the loop from digging into the agar. 5. Remove the loop and close the lid. 6. Resterilize the inoculating loop. (Pour plate: Add the inoculum then pour the sterile molten and cooled agar for preparation of pour plates of plates for Spread plate, the inoculum is spread on the solidified agar surface of the plate with a L- shaped spreader.)

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Introduction to the Basic Microbiology Laboratory Practices

(a) Fig. 3.2: Streaking on agar plate

(b)

C. Transferring the inoculum into an agar slant or agar stab 1. Remove the cap or plug of the tube still holding within the fingers of the hand. 2. Insert the loop carrying the scraped inoculum into the tube. 3. Streak along the surface of the slant with the loop. In case of agar stab, stab the agar with the needle as shown. 4. Incubate the inoculated tube, agar slant, agar deep tube or agar plates at the optimum growth temperature of the inoculated cultures. 5. Observe the various growth patterns.

Fig. 3.3: Sub-culturing into agar slant and agar stab

3.6

OBSERVATIONS

After incubation, check the following growth patterns of all tubes and plates. Note down the observations in the given table. 19

Practical Manual Food Microbiology

Fig. 3.3: Microbial growth pattern on agar slant, agar stab and agar plates

V. 3.7
1.

RESULTS

Draw and describe the growth seen in each of the four given broth cultures.

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Introduction to the Basic Microbiology Laboratory Practices

A Growth is = 2.

B Growth is =

C Growth is =

D Growth is =

Observe the growth in the slant cultures and stab cultures for pigmentation and purity. Using the terms mentioned above compare a single colony of culture A with a single colony of culture B. Use a hand lens or a dissecting microscope to magnify the colony. Characteristics Form of colony Elevation Margin (edge) Surface Optical characteristics Pigmentation A B

3.

VI. 3.8

PRECAUTIONS

1. Glassware should be properly cleaned and sterilized. 2. Weighing should be done accurately. 3. pH adjustment should be done carefully. 4. Filtering should be proper. 5. Cotton plugs should be tight enough. 6. Never lay the cap down or it may become contaminated 7. Allow the loop to cool a few seconds to avoid killing the microbes in inoculum.

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