25 September 2016

Lecture #2: CULTIVATION OF BACTERIA





Propagating the microorganisms under laboratory condition is known as cultivation. To accomplish this, one must know the:

a.      Food nutrients required
b.      Proper environmental conditions

Nutritional requirements

1.      Carbon source

Autotrophs or lithotrophs  - require only CO2 for their carbon source.

Heterotrophs or organotrophs – require and organic form of carbon

a.      Saprophytes – survive in dead organic matter.
b.      Parasites – require living organism for life

2.      Nitrogen source

Some uses atmospheric nitrogen, some thrive on inorganic nitrogen compounds and others derive their nitrogen from proteins or any naturally occurring organic nitrogen compounds.

3.      Minerals

In the form of organic sulfur and free inorganic phosphates

4.      Metallic elements

Enzyme activators like Mg, Fe, Na, K, Mn, Zn, Cu and Co.

5.      Growth factors like vitamins and amino acids

6.      Water


Physical conditions

1.      Temperature requirements

On the basis of temperature requirement, bacteria are classified as:

a.      Psychrophiles or cryophiles (cold–loving) – grow at 20oC or less

b.      Mesophiles (moderate–loving) – grow at 25oC–40oC

c.       Thermophiles (heat–loving) – grown between 45oC–60oC

Facultative thermophiles or eurithermophiles – these are thermophilic bacteria that extends into the mesophilic region

Stentothermophilic – thermophilic bacteria that grows above 60oC.

2.      Gaseous requirements

The principal gases that affect bacterial growth are oxygen and carbon dioxide. Based on oxygen requirement, bacteria are divided into:

a.      Aerobic – grows in the presence of free oxygen

b.      Anaerobic – grows in the absence of free oxygen

c.       Facultatively anaerobic – grown in either absence or presence of free oxygen

d.     Microaerophilic – grows in the presence of minute quantities of free oxygen.

3.      pH or Hydrogen ion concentration

This is the degree of acidity or alkalinity. For most bacteria, the optimum pH for growth lies between 6.5 and 7.5. Some grow as low as pH 3 like the fungi and others grow as high as pH 10.5 like the vibrios

4.      Miscellaneous physical requirement

a.      Source of energy

Photosynthetic autotrophic – requires light as source of energy

b.      Osmotic or hydrostatic pressure

Osmophilic bacteria – requiring high osmotic pressure
Others are isolated from the deepest ocean trenches

c.       High concentration of salt

Halophilic bacteria


THE CULTURE MEDIA

A culture media is any material containing essential nutrients for the growth and multiplication of bacteria. Practically, all media are available commercially in powdered form.

Types of culture media

1.      According to physical state / consistency

a.      Liquid – contains no agar, no gelatin

e.g. Thioglycollate, Trypticase Soy Broth

b.      Solid – with agar, gelatin or albumin

Agar – is a polysaccharide derivative of seaweed, dissolves in water at boiling point and set at 38oC. It is a solidifying agent with a concentration of 1.5 to 3%.

Gelatin – used as a diagnostic medium and solidifies at 25oC and has 10 to 15% concentration

Albumin – coagulating factor of the agar

e.g. Blood Agar Plate, Eosin Methylene Blue, TSI, LIA

c.       Semi–solid – contains 0.5 to 1% agar. It is used to demonstrate “swarming” growth of some species of Proteus. Also used in motility test.
e.g. SIM

2.      According to application of function

a.      Simple, ordinary or basal – basic medium form which culture media are prepared and supports the growth of many common bacteria

b.      Enriched – prepared so as to duplicate the natural environment of the desired bacteria or fastidious heterotrophs that are difficult to grow.

e.g. Blood Agar Plate, Chocolate Agar Plate, Loeffler’s serum agar

c.       Differential – differentiates two or more species of microorganisms by their colony characteristics on the same medium due to the dye or indicator incorporated in the medium

Eosin Methylene Blue agar – eosin and methylene blue
                  Mannitol Salt agar – phenol red
                  Simmon Citrate agar – bromthymol blue
                  McConkey agar – neutral red

d.     Selective – allows the growth of some bacteria while preventing or suppressing the growth of others because of inhibitory substances in the medium.

Inhibitory substances                           Inhibits

Gentian Violet                                        gram positive bacteria
Sodium Desoxycholate
Bile salts

Chloral hydrate                                      spreading of Proteus
Alcohol

Sodium azide                                         gram negative bacteria
Potassium tellurite                               

Penicillin / Streptomycin                      bacterial invaders or contaminants
Malachite Green

e.g. Salmonella–Shigella agar, Petragnanni medium, Mannitol Salt agar,
                  Saboraud’s agar

3.      According to composition

a.      Synthetic or chemically defined – pure chemicals which substitute the requirement derived from natural sources.

e.g. Glucose – inorganic salts – for E. coli

b.      Non–synthetic – exact composition is not known.

e.g. Commercially prepared media

c.       Tissue culture – made of living cells

e.g. HeLa cell lines

4.      According to form or distribution

a.      Plated medium – placed on a petri dish
b.      Tubed medium – placed on a test tube

Preparation of culture media

1.      Weighing of different ingredients and placing appropriate container
2.      Dissolving the material in proper amount of distilled water
3.      Titration – adjusting to right pH
4.      Sterilization
5.      Distribution on sterile petri dishes
·         For tubed method – distribute before sterilize
·         Inside refrigerator – normal position (cover is on top)
·         Inside incubator – inverted position, to prevent evaporation


Aerobic cultivation of large quantities

1.      Use of suitable containers such as Kolle flask, Roux bottle and Fernbach flask.
2.      Constant shaking of liquid medium to introduce oxygen.
3.      Forcing air through the medium by pressure or suction.

Anaerobic cultivation

1.      Addition of reducing agent such as sodium thioglycollate

2.      Mechanical removal of oxygen from an enclosed vessel, by pumping the air out and replacing it with nitrogen, hydrogen or H2–CO2 mixture (GasPak).


3.      Sealing tubes of agar with a layer of petrolatum, oil or paraffin

4.      Chemical reaction within an enclosed vessel containing the inoculated medium to combine free O2 into a compound by combustion:

a.      Burning of a candle – Candle Jar method
b.      Use of Brewer anaerobic jar

5.      Chemical reaction using a mixture of 40% pyrogallic acid and NaOH

6.      Use of enriched agar in deep tubes

Containers with bacteria are placed in an incubator, an apparatus in which constant degree of temperature (37oC) is maintained for purpose of growing cultures of bacteria.

Control of culture media

1.      Follow exact directions of the manufacturer regarding methods of preparation and sterilization (overheating is the most frequent source of error).

2.      Sealing and dating properly all dehydrated media

3.      Check the pH of the final product

4.      Freshly prepared media are preferable for anaerobic bacteriology

Quality control program is best carried out by performance testing for the desired reaction with a number of stock cultures of known microorganisms of known stability.

Stock culture media may be safely stored for months if care is taken to retain moisture.

Transport media

1.      Amies transport medium with charcoal – maintenance of fastidious organisms in original specimen
2.      Carry and Blair – for feces
3.      Stuart’s

THE PROCESS OF INOCULATION

Inoculation is a process of implanting microbes or infectious material into a culture media. Cultures are growth of microorganisms on nutrient medium while culture means to grow the microorganisms on such medium.

Types of cultures:

1.      Pure cultures – when microorganism in a culture medium is all of the same species.

2.      Mixed cultures – when there are two or more different species of microorganism growing in a culture medium.

3.      Contaminated cultures – when the cultures accidentally contain more than one species of microorganisms.

4.      Stock culture – is a pure culture of microorganisms used as a source of supply for research, industry or student use.

Manner of inoculating a culture medium:

A culture medium is inoculated by streaking or swabbing gently a previously sterilized swab or platinum loop or needle containing such as microbes, sputum, urine, blood or pus.

a.      Petri dish containing the culture medium – by streaking using either:

(1)   Simple streaking
(2)   Radial method
(3)   Clock method

b.      Butt culture medium – by stabbing

Using a previously sterilized needle, stab the butt medium at the center. The portal of entry of the needle should be the same as the portal of exit.

c.       Butt–slant culture medium – stab and streak

Using a previously sterilized needle, the center of the butt portion is stabbed, withdraw the needle and streak in a “zigzag” motion up to the upper portion of the slant.

d.     Slant culture medium – streak

This is inoculated by streaking the surface of the slant with a previously sterilized loop with a “zigzag” motion up to the upper portion of the slant.

e.      Liquid medium – incline the tube at an angle and rub the material against the tube wall.

Inoculating loop or needle should be flame–sterilized before and after using it. The neck of the tube or bottle of culture should be flame–sterilized before and after re – capping or replacing the cap.

In incubation, petri dishes containing the inoculated agar should be placed inside the incubator in an inverted position to prevent the accumulation of moisture in the cover to drop into the medium and destroy colonies.

Methods employed in obtaining a pure culture:

1.      Streak plate method – surface streaking

The routine procedure for isolating bacteria in a pure culture.


2.      Pour plate technique

The dilution (thinning) of the specimen in tubes of liquid (cooled) agar medium. The streak plate and pour–plate techniques can be made more effective for isolating specific kinds of bacteria by using selective or differential media.


3.      Enrichment – culture technique

It provides a specially designed cultural environment which will favor the growth of the particular type of bacteria being sought but will be unsuitable for the growth of other types.

4.      Serial dilution technique


Series of dilutions in tubes of an appropriate medium containing decreasing number of bacteria.

5.      Single–cell isolation technique

This technique uses a micromanipulator to pick out a single organism from a hanging drop preparation.

Pure cultures should be maintained and preserved by:

a.      Periodic transfer to fresh media
b.      Overlaying cultures with mineral oil
c.       Lyophilization – drying in a frozen state
d.     Storage at a very low temperature

Cultural characteristics

One of the major features of bacteria is their appearance following growth on various media. Such commonplace characteristics as the color, abundance of growth and even the odor of the culture provide useful clues for identification.

1.      Agar plate colonies

a.      Size – colonies range in size from extremely small (pinpoint) to large colonies. Many form a colony of a limited size regardless of the period of incubation. Others, like Pseudomonas and Proteus spread across the entire agar surface.

b.      Margin or edge – it may be evenly circular or it may show irregularities as rounded projections, irregular notches, threadlike or rootlike projections.

c.       Elevation – colonies may either be flat or raised.

d.     Chromogenesis or pigmentation – colonies may be colored or not.

e.      Optical features – maybe opaque, translucent or opalescent


2.      Growth on agar slant

a.      Amount – scanty, moderate or abundant

b.      Margin or edge of growth – similar to agar plat

c.       Consistency of mass growth – butyrous or butterlike consistency, easily removed with transfer needle; viscous or stingly; dry and brittle.

d.     Chromogenesis or pigmentation – similar to that described for colonies.

3.      Growth on nutrient broth

a.      Amount – scanty, abundant or moderate

b.      Distribution or growth – evenly turbid; growth confirmed to surface of broth as a scum or film (pellicle); or growth accumulated as sediment, which may be granular or viscous.

c.       Odor – may be putrid, fruity or aromatic or negligible

4.      Growth in gelatin stabs

a.      Growth (no liquefaction) along line of inoculation – growth may be confined to zone of inoculation streak or may exhibit varying degrees of spreading away from this streak.

b.      Liquefaction of gelation – may start evenly from top or various designs of liquefied medium (funnel–like) may occur.

Chromogenesis is one of the most striking cultural characteristics. Not all bacterial species have this distinctive feature. Not all bacterial species have these distinctive features. In some, the pigment is retained within the cell and the mass of bacterial cells is colored; others the pigment is excreted and colors the medium.

The intensity of the pigment is influenced by the comparison of the medium and the conditions of incubation. Pigment production is best observed from growth on solid media.

Enrichment broth:

1.      Alkaline peptone water                  Vibrio cholera
2.      Selenite F broth                                Salmonella and Shigella
3.      Tetrathionate broth                          Salmonella except S. typhi
4.      Gram negative broth                       Salmonella and Shigella in feces

Ringer’s solution – for dissolving calcium alginate swab


                        CULTURE MEDIA                                                 PURPOSE  

            Blood Agar Plate                              cultivation of fastidious organisms;
determination of hemolytic reactions
           
            Brain Heart Infusion Agar              cultivating the pneumococcus for the liquid
                                                                        bile solubility test

            Brilliant Green Agar                        highly selective for the isolation of Salmonella

            Brucella–Vit K1 Blood Agar          for the isolation and subculture of anaerobes

            Campy–blood agar                          selective for the isolation of Campylobacter

            Buffered Charcoal Yeast Extract    selective for Legionella sp.

            CDC anaerobic agar                         isolation of anaerobic organisms

            Cefsulodin–Irgasan–
                        Novobiocin Agar                 selective for Yersinia sp.

            Chocolate Agar                                 cultivation of Haemophilus and Neisseria

            Columbia Colistin–Nalidixic
                        Acid agar (CAN)                  selective for isolation of gram positive cocci

            Cycloserine–cefoxitin fructose       selective for Clostridium difficile
                        Agar (CCFA)

            Cystine–lactose electrolyte                         isolation and enumeration of bacteria in
                        Deficient Agar (CLED)        urine

            Cytine Tellurite Blood Agar          isolation of C. diptheriae

            Decarboxylase test media               for differentiating members of the
Enterobacteriacea

            Desoxycholate Agar                                    for the isolation of gram negative enteric           
                                                                        Bacilli and the differentiation of lactose–
                                                                        fermenting and non–lactose fermenting
                                                                        species

            DNA agar                                          for testing S. aureus for thermostable nuclease

            Dextrose Ascitic fluid
                        Semisolid agar                      for spinal fluid cultures

            Ellinghausen, McCullough,
            Johnson and Harris (EMJH)            cultivation of leptospires
                        medium

            Fetal Bovine Serum agar with        isolation of H. ducreyi
                        Vancomycin

            Gelatin medium (dilute)                 differentiation of Nocardia and Streptomyces

            Hektoen Enteric Agar (HEA)         for isolation and differentiation of gram
                                                                        negative enteric pathogens; coliforms are
                                                                        salmon to orange in color; salmonella and
                                                                        shigella are bluish green

            Hemin solution                                as a supplement for recovery of Hemophilus
                                                                        species from respiratory culture

            Horse blood–bacitracin agar          as a selective medium for Hemophilus species

            Human Blood Bilayer (HBB)         selective and differential isolation of
                                                                        Gardnerella vaginalis

            Kanamycin–Vancomycin                for primary inoculation of clinical specimens
            Blood Agar (KVBA)                         for selective isolation of anaerobes, particularly
            Bacteroides

            Kelly’s medium                               non–selective modified for the isolation of
                                                                        B. burgdorferi & other spirochetes

            Lombard–Dowell agar                    isolation of anaerobic organisms

            Lysine Iron Agar                              for determining whether members of the
                                                                        Family Enterobacteriacea can decarboxylate
                                                                        or deaminate lysine

            MacConkey agar                              Inhibitory for gram positive bacteria and
                                                                        differential for enteric bacteria rather than         
                                                                        selective

            McBride medium                             cultivation of Listeria

            Middlebrook 7H10 agar                 isolation of and antimicrobial susceptibility     
                                                                                    testing of Mycobacteria

            New York City (NYC) agar            selective for Neisseria gonorrhea and
                                                                        M. hominis

            Methylene Blue milk                       identification of enterococci

            Milk media – Litmus milk             determination of litmus milk reactions of
                                                                        Clostridium

            Mueller Hinton Agar                       Useful for the disk agar diffusion method of
                                                                        testing for antimicrobial susceptibility testing

            Oxidative–Fermentative (O–F)      differentiation of Pseudomonas, Alcaligenes,
                        Basal medium                      Acinetobacter from Enterobacteriacea

            Peptic Digest Agar                           a good medium for H. influenza

            Phenylethanol Agar (PEA)             isolation of gram positive cocci and inhibition
                                                                        of gram negative bacilli particularly Proteus

            Schaedler Agar                                 Nonselective medium for the recovery of
                                                                        anaerobes and aerobes

            Scott’s modified Castaneda            For blood cultures
            Media and Thioglycollate broth   

            Seller’s differential agar                  for differentiating and identifying
                                                                        Nonfermentative gram (–) bacilli that produce
                                                                        an alkaline reaction on TSI agar; useful in
                                                                        differentiating Pseudomonas aeruginosa,
                                                                        Acinetobacter calcoaceticus and Alcaligenes
                                                                        fecalis

            Sodium bicarbonate                                    supplement for anaerobes

            Sodium chloride broth                    selective for enterococci and salt tolerant
                                                                        organisms

            Streptococcal selective agar           selective for Streptococcus pyogenes

            Thioglycollate medium                  recovery of anaerobes, aerobes,
                        without indicator                 microaerophilic & fastidious organisms

            Tinsdale agar                                                isolation of C. diphtheria

            Todd–Hewitt broth                          for growing streptococci for serologic
                                                                        identification

            Trypticase soy agar                                     Excellent blood agar base and can be used
                                                                        For the isolation and maintenance of all
                                                                        organisms

            Trypticase soy broth                        supports growth of pneumococci and
                                                                        Streptococci and Brucella

            Tween 80–albumin broth                cultivation of spirochetes

Xylose Lysine Desoxycholate        isolation & differentiation of Salmonella
(XLD) agar                                         & Shigella agar from other enteric bacilli
                                               





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