MICROBIOLOGY : BACTERIA

STRUCTURE OF BACTERIA

SIZE :

Diameter : Bacteria are very small, most being approximately 0.5 to 1.0 µm in diameter.

Surface area : around 12 µm.

Volume: 4 µm.

SHAPE : The particular shape is not yet understood. Typical bacterial cells are

1. spherical ( cocci ; singular, coccus)

2. straight rods ( bacilli ; singular bacillus)

3. rods that are helically curved ( spirilla ; singular, spirillum)

4. curved ( vibrio)

STRUCTURE :

Bacteria’s main component

1. flagella

2. cytoplasm

3. Cell membrane

4. capsule

5. cell wall

1. FLAGELLA

Bacterial flagella ( singular, flagellum )are hair like, helical appendages that protrude through the cell wall and are responsible for swimming motility. They are much thinner , being 0.01 to 0.02 µm in diameter. A flagellum composed of three parts : a basal body, a short hook and a helical filament which is usually several times as long as the cell.

According to arrangement of flagella bacteria are of four types :

a) Monotrichous; a single polar flagellum.

b) Lophotrichous; a cluster of polar flagella

c) Amphitrichous; flagella, either single or clusters, at both cell poles

d) Peritrichous; surrounded by lateral flagella .

2. Cytoplasm

i) cytoplasm consists of 80% water & 20% salt & protein.

ii) bacterial DNA is circular & haploid

iii) bacteria has extra circular DNA known as plasmid

iv) it has no organelles like mitochondria, Golgi complex.

Function of cytoplasm :

I) control osmotic pressure

II) DNA is haploid for

-rapid production & protein synthesis

III) plasmid has the function to resist antibiotic by some enzyme,e.g. ß-lactamase.

3. CELL MEMBRANE :

I) It is a phospholipid by layer.

II) Water can penetrate through it

III) It is flexible & not strong

IV) it can be ruptured easily by the osmotic pressure created within cytoplasm.

4. CAPSULE:

Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around the cell wall. If this layer can be visualized by light microscopy using special staining methods, it is termed a capsule.

If the layer is too thin to be seen by light microscopy it is termed a microcapsule.

FUNCTIONS OF CAPSULE:

Capsules can serve a number of functions, depending on the bacterial species:

(1) They may provide protection against temporary drying by binding water molecules.

(2) They may block attachment of bacteriophages.

(3) They may be antiphagocytic; i.e., they inhibit the engulfment of pathogenic bacteria by white blood cells and thus contribute to invasive or Infective ability (virulence).

(4) They may promote attachment of bacteria to surfaces; for example, Streptococ­cus mutans, a bacterium associated with producing dental caries, firmly adheres to the smooth surfaces of teeth because of its secretion of a water-insoluble capsular glucan.

(5) If capsules are composed of compounds having an electri­cal charge, such as sugar—uronic acids, they may promote the stability of bac­terial suspension by preventing the cells from aggregating and settling out.

COMPOSITION:

Most bacterial capsules are composed of polysaccharides.

Homopolysaccharides: Capsules composed of a single kind of sugar, usually synthesized outside the cell. e.g. the synthesis of glucan from sucrose by S. mutans.

Heteropolysaccharides: Other capsules are composed of several kinds of sugars, usually synthesized from sugar precursors that are activated within the cell and polymerized outside the cell. The capsule of Klebsiella pneumoniae is an example.

NUTRITIONAL REQUIREMENTS FOR BACTERIAL GROWTH :

All forms of life, from microorganisms to human beings, share certain nutritional requirements for growth and normal functioning. The following observations:

1 All organisms require a source of energy. Some rely on chemical compounds for their energy and are designated as chemotrophs.^. Others can utilize radiant energy (light) and are called phototrophs.

2 All organisms require a source of electrons for their metabolism. Some organisms can use reduced inorganic compounds as electron donors and are termed lithotrophs (some may be chemolithotrophs others photochemolithotrophs). Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs, others photoorganotrophs).

3 All organisms require carbon in some form for use in synthesizing cell components.

some bacteria can use CO₂ as their major source of carbon; such organisms are termed autotrophs. Others require organic compounds as their carbon source and are termed heterotrophs

4 All organisms require nitrogen in some form for cell components. Bacteria are extremely versatile in this respect. Unlike eukaryotes, some bacteria can use atmospheric nitrogen. Others thrive on inorganic nitrogen compounds such as nitrates, nitrites, or ammonium salts, and still others derive nitrogen from organic compounds such as amino acids.

5 All organisms require oxygen, sulfur and phosphorus for cell components.

6 All living organisms require metal ions, such as K⁺, Ca²⁺, Mg²⁺ and Fe²⁺ for normal growth. Other metal ions are also needed but usually only at very low concentrations, such as Zn²⁺_, Cu²⁺, Mn²⁺ _, Mo⁶_, Ni²⁺ , B³⁺, and CO²⁺ ; these are often termed trace elements and support bacterial growth.

7 All living organisms contain vitamins and vitaminlike compounds Some bacteria are capable of synthesizing their entire requirement of vitamins from other compounds in the culture medium.

8 All living organisms require water and in the case of bacteria all nutrients must be in aqueous solution before they can enter the cells.

CLASSIFICATION OF BACTERIA :

A) According to P^H requirement :

I) Acidophiles : identified in acid medium(P^H range: 1to 5.50)

II) Basophiles : identified in basic medium(P^H range: 8.5 to 11.5)

III) Neutrophiles : identified in slight acid and slight medium (P^H range:5.5 to 8)

B) According to energy sources :

I) Autotrophs : energy source is inorganic ions, metal

II) Heterotrophs : energy source is organic, carbohydrate

C) According to Oxygen requirement :

I) Obligatory aerobe ( oxygen is must required )

II) Facultative aerobe

III) Anaerobe ( no oxygen is required )

IV) Obligatory anaerobe

BACTERIOLOGICAL MEDIA:

Certain raw materials such as peptone, meat extract, and yeast extract are used to prepare bacterial media. Agar may be added to this media if solidification is required.

PREPARATION OF MEDIA:

The preparation of bacteriological media usually involves the following steps:

1 Each ingredient, or the complete dehydrated medium, is dissolved in the appro­priate volume of distilled water.

2 The pH of the fluid medium is determined with a pH meter and adjusted if necessary.

3 If a solid medium is desired, agar is added and the medium is boiled to dissolve the agar.

4 The medium is dispensed into tubes or flasks.

5 The medium is sterilized, generally by autoclaving. Some media (or specific ingredients) that are heat-labile are sterilized by filtration.

Types of Media: On the basis of application or function, media may be classified as follows.

Selective media:

These media provide nutrients that enhance the growth and predominance of a particular type of bacterium and do not enhance (and may even inhibit) other types of organisms that may be present.For instance, a medium in which cellulose is the only carbon source enrich the growth of cellulose-utilizing organisms when it is inoculated with a soil sample containing many kinds of bacteria. As an example of a different type of selective medium, the isolation of the gonorrhea-causing organism,Neisseria gonor­rhoeae, from a clinical specimen is facilitated by the use of media containing certain antibiotics;these antibiotics do not affect N. gonor­rhoeae but do inhibit the growth of contaminating bacteria.

Differential media:

Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood-containing agar medium (blood agar), some of the bacteria may hemolyze (destroy) the red blood cells; others do not. Thus one can distinguish between hemolytic and nonhemolytic bacteria on the same medium.

Media for Enumeration of Bacteria:

Specific kinds of media are used for determining the bacterial content of such materials as milk and water. Their composition must adhere to prescribed spec­ifications.

Maintenance Media:

Satisfactory maintenance of the viability and physiological characteristics of a culture over time may require a medium different from that which is optimum for growth. Prolific, rapid growth may also be associated with rapid death of the cells at the end of the growth phase. For example, glucose in a medium frequently enhances growth, but acid harmful to the cells is likely to be pro­duced. Therefore, omission of the glucose is preferable in a maintenance me­dium.

Types of media: On the basis of physical state media may be classified as follows.

Liquid media:

It is water based solution & it does not solidify at temperature above freezing point. Thus it is free flowing media.

In addition to liquid media, solid and semisolid media are widely used for cultivation of bacteria.

Solid media:

Solid media are useful for isolating bacteria or for deter­mining the characteristics of colonies. The solidifying agent is usually agar. Silica gel is sometimes used as an inorganic solidifying agent for autotrophic bacteria.

Semisolid media:

Semisolid media, prepared with agar at concentrations of 0.5 percent or less, have a soft, custard like consistency and are useful for the cultivation of mi­croaerophilic bacteria or for determination of bacterial motility.

PHYSICAL CONDITIONS REQUIRED FOR GROWTH:

1 Temperature:

Since all processes of growth are dependent on chemical reactions and since the rates of these reactions are influenced by temperature:

On the basis of their temperature relationships, bacteria are divided into three main groups:

Psychrophiles:

Psychrophiles are able to grow at 0°C or lower, but

have an optimum temperature of 15°C or lower 'and

a maximum temperature of about 20°C.

Mesophiles :

Grow best within a temperature range of approximately 25 to 40° C. e.g. all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing best at about body temperature (37°C)

Thermophiles :

Grow best at temperatures above 45°C.

2 Gaseous Requirements:

The principal gases that affect bacterial growth are oxygen and carbon dioxide. Bacteria display such a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases:

Aerobic bacteria: require oxygen for growth and can grow when incubated in an air atmosphere (i.e., 21 percent oxygen).

Anaerobic bacteria : do not use oxygen to obtain energy; moreover, oxygen is toxic for them and they cannot grow when incubated in an air atmosphere

Facultatively anaerobic bacteria: do not require oxygen for growth, although they may use it for energy production if it is available.

Microaerophilic bacteria: require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere.

3 P^H requirement:

For most bacteria the optimum P^H for growth lies between 6.5 and 7.5, and the limits generally lie somewhere between 5 and 9. However, a few bacteria prefer more extreme P^H values for growth. e.g.

Thiobacillus thiooxidans grow in a range between P^H 0.5 to 6.0.

An unclassified bacteria in California spring grow best at a P^H of 9.0 to 9.5.

GROWTH : BINARY FISSIONS

(How can we calculate the number of generation from the final count of bacteria?)

The most common means of bacterial reproduction is binary fission; one cell divides, producing two cells. Thus, if we start with a single bacterium, the increase in population is by geometric progression:

1 → 2 → 2² → 2³ → 2⁴ → 2⁵ …... 2ⁿ

where n = the number of generations. Each succeeding generation, assuming no cell death, doubles the population. The total population N at the end of a given time period would be expressed

N = 1 x 2ⁿ ………………….(1)

However, under practical conditions, the number of bacteria N_o inoculated at time zero is not 1 but more likely several thousand , so the formula now becomes

N = N_o X 2ⁿ (2)

Solving Eq. (2) for n, we have

log₁₀N = log₁₀ N_o + n log₁₀2

n = log₁₀ N — log₁₀ N₀ / log_1o2……….(3)

If we now substitute the value of log_1o 2, which is 0.301, in the above equation, we can simplify the equation to

n = Log₁o N — log_1o N_o / 0.301

n = 3.3 (log_IO N — log_1O N_O)…………(4)

Thus, by use of Eq. (4), we can calculate the number of generations that have taken place, providing we know the initial population and the population after growth has occurred.

Explain Bacterial Growth Curve.

Lag phase:

In this stationary phase the bacteria are not quiescent or dormant rather they collect nutrients from the media. During this stage the individual cells increase in size beyond their normal dimensions. Different enzymes are required for this function & bacteria synthesize this enzymes during this period. At the end of the lag phase, each organism divides.

The logarithmic or Exponential phase:

During this period the cells divide steadily at a constant rate, and the log of the number of cells plotted against time results in a straight line. Thus the straight line indicating rapid growth is observed. The population is most nearly uniform in terms of chemical composition of cells, metabolic activity, and other physiological characteristics.

The stationary phase:

The population remains constant for a time. The amount of nutrient given into the media begin to deplete and toxic products are produced which block the division or causes death of some bacterial cells. Thus the reproduction rate is balanced by an equivalent death rate.

The phase of decline or death:

Following the stationary phase the bacteria may die faster than new cells are produced, if indeed some cells are still reproducing. During the death phase, the number of viable cells decreases exponentially , essentially the inverse of growth during the log phase.

quantitave measurement of BACTERIAL GROWTH:

We have seen that the term growth as commonly applied in microbiology refers to the magnitude of the total population. Growth in this sense can be determined by numerous techniques based on one or more of the following types of measurement:

1 Cell count. Directly by microscopy or by using an electronic particle counter, or indirectly by a colony count

2 Cell mass. Directly by weighing or by a measurement of cell nitrogen, or indirectly by turbidity

3 Cell activity. Indirectly by relating the degree of biochemical activity to the size of the population

DIRECT MICROSCOPIC COUNT:

Bacteria can be counted easily and accurately with the Petroff-Hausser counting chamber. This is a special slide accurately ruled into squares that are 1/400 mm² in area; a glass cover slip rests 1/50 mm above the slide, so that the volume over a square is 1/20,000 mm³ (1/20,000,000 cm³). A suspension of unstained bacteria can be counted in the chamber, using a phase-contrast microscope. If, for example, an average of five bacteria is present in each ruled square, there are 5 X 20,000,000, or 10⁸, bacteria per milliliter.

ADVANTAGES:

1.Direct microscopic counts can be made rapidly and simply with a minimum of equipment

2.The morphology of the bacteria can be observed as they are counted.

3. Very dense suspensions can be counted if they are diluted appropriately.

DISADVANTAGES:

1.suspen­sions having low numbers of bacteria, e.g., at the beginning of a growth curve, cannot be counted accurately.

2. There is no way to determine whether the cells being counted are viable.

ELECTRONIC ENUMERATION OF CELL NUMBERS:

In this method, the bacterial suspension is placed inside an electronic particle counter, within which the bacteria are passed through a tiny orifice 10 to 30 µm in diameter. This orifice connects the two compartments of the counter which contain an electrically conductive solution. As each bacterium passes through the orifice, the electrical resistance between the two compartments increases momentarily. This generates an electrical signal which is automatically counted.

ADVANTAGES:

Accurate and rapid method as it requires sophisticated electronic equipment;

DISADVANTAGES:

1.The orifice tends to become clogged if a large or joined bacteria can pass.

2.There is no way to determine whether the cells being counted are viable.

PLATE COUNT METHOD

This method, illustrated in Fig, allows determination of the number cells that will multiply under certain defined conditions. A measured amount of the bacterial suspension is introduced into a Petri dish, after which the agar medium (maintained in liquid form at 45°C) is added and the two thoroughly mixed by rotating the plate. When the medium solidifies, the organisms are trapped in the gel. Each organism grows, reproducing itself until a visible mass of organisms—a colony—develops; i.e., one organism gives rise to one colony. Hence, a colony count performed on the plate reveals the viable microbial population of the inoculum. The original sample is usually diluted so that the number of colonies developing on the plate will fall in the range of 30 to 300. Within this range the count can be accurate, and the possibility of interference of the growth of one organism with that of another is minimized. Colonies are usually counted by illuminating them from below (dark-field illumination) that they are easily visible, and a large magnifying lens is often used. Various electronic techniques have been developed for the counting of colonies.

ADVANTAGES:

1. It is useful for counting in milk, water, food or other materials.

2. Easy to perform

3. Small numbers can also be counted

DISADVANTAGES:

1 The bacterial suspension must be free of aggregates & the mixing should be homogenous.

2. The medium can only be used if that is suitable for a specific bacteria.

Turbidimetric Methods:

Bacteria in a suspension absorb and scatter the light passing through them, so that a culture of more than 10⁷ to 10⁸ cells per milliliter appears turbid to the naked eye. A spectrophotometer or colorimeter can be used for turbidimetric measurements of cell mass.

ADVANTAGES:

1. Turbidimetry is a simple, rapid method

DISADVANTAGES:

1. The culture must be dense enough to register some turbidity on the instrument.

2. It may not be possible to measure cultures grown in deeply colored media or cultures that contain suspended material other than bacteria.

PURE MICROBIAL POPULATION

CHEMICAL METHODS OF SELECTION

1.USE OF A SPECIAL CARBON OR NITROGEN SOURCE:

One type of chemical method is to provide in the culture medium a substrate, i.e., a single carbon or nitrogen source, that can be used only by the species being sought (Fig.). This particular kin of selection is often referred to by a special name, enrichment. For example, if we wish to isolate, from soil, bacteria capable of utilizing a very complex organic compound like a-conidendrin a constituent of wood, we find that when we inoculate a medium such as nutrient agar directly with the soil sample, our chances of finding a-coniden­drin-utilizng bacteria will be very limited. There are so many other rapidly growing bacteria present that the more slowly growing kind we wish to obtain will be soon overgrown. Consequently, we prepare a liquid enrichment medium in which a-conidendrin is the sole source of carbon. Under these conditions only organisms capable of utilizing this compound will be able to grow well. However, it is important to recognize that other bacteria may be able to grow to some extent by utilizing organic compounds made by the conidendrin-utilizing organisms and that the method is not completely specific. As another example, if we wish to select for nitrogen-fixing bacteria, nitrogen gas _(N2) can be supplied as the sole nitrogen source, since only nitrogen-fixing bacteria will be able to grow well. Other bacteria may grow, but to a lesser degree, by using the nitrog­enous products made by the nitrogen-fixers.

2. USE OF DILUTE MEDIA:

Certain aquatic bacteria, such as Caulobacter species, are capable of growing with very low levels of carbon or nitrogen sources. Consequently, one way to select for such bacteria is to inoculate a mixed culture into a very dilute medium, e.g., a broth containing only 0.01 percent peptone. peptone. The medium must have low enough levels of nutrients that other kinds of organisms will not be able to grow well in it.

3. USE OF INHIBITORY OR TOXIC CHEMICALS:

The addition of low levels of certain chemicals, such as dyes, bile salts,salts of heavy metals, or antibiotics,to culture media can be useful for the selection of certain kinds of bacteria. The following are examples of this type of selection:

a) Many Gram-negative bacteria can grow in the presence of low concentrations of various dyes that inhibit the growth of Gram-positive bacteria. Similarly, intestinal bacteria can grow in the presence of bile salts such as sodium deoxycholate, whereas nonintestinal bacteria are usually inhibited. Consequently, a medium containing crystal violet dye plus sodium deoxycholate will allow Gram-negative intestinal bacteria to grow but will inhibit most other kinds of bacteria. An example of such a medium is MacConkey agar, which is widely used to select for Gram-negative intestinal pathogens such as Salmonella and Shigella species.

b) Campylobacter jejuni is a frequent cause of diarrhea in humans, yet

diarrheic stool samples contain many other kinds of bacteria that interfere with the isolation of this species. By incorporating certain antibiotics or other chemotherapeutic agents, such as vancomycin, polymyxin, and trimethoprim, into the culture me­dium, most of these contaminants can be inhibited without affecting the growth of C. jejuni.

PHYSICAL METHODS OF SELECTION

1. HEAT TREATMENT:

To select for endospore-forming bacteria, a mixed culture can be heated to 80°C for 10 min before being used to inoculate culture media. Vegetative cells will be killed Cy this treatment but endospores will survive and subsequently germinate and grow.

2. INCUBATION TEMPERATURE:

To select for psychrophilic or psychrotrophic bacteria, cultures are incubated at low temperatures, e.g., 0 to 5°C. For selection of thermophiles, a high incu­bation temperature is used, e.g., 55°C.

3. P^H OF THE MEDIUM:

To select for acid-tolerant bacteria, a low-pH medium can be used. For example, to select for the lactobacilli present in cheddar cheese, the pH of the medium is maintained at 5.35 with an acetic acid/acetate buffer; other organisms in the cheese cannot grow well at such a low pH. Similarly, to select for alkali-tolerant organisms, a high-pH medium can be used. For example, to select for the cholera-causing bacterium, Vibrio cholerae, from a stool sample, we can use a medium with a pH of 8.5; most other intestinal bacteria are unable to grow at this pH.

4. Cell Size and Motility:

We can sometimes make use of a small cell diameter or of bacterial motility to achieve selection. For instance, Treponema species from the human oral cavity can be selected by taking advantage of both of these properties. A membrane filter having a pore size of 0.15 µm is placed on the surface of an agar plate and gingival scrapings are placed on the filter. The unusually small size of treponemes allows them to penetrate the pores of the filter to reach the underlying agar. Moreover, treponemes have the ability to swim through solid agar media; consequently, they migrate away from the filter and grow to form a hazy zone within the agar, from which they can be subcultured. Other bacteria from the oral cavity are either too large to penetrate the membrane filter or, if they can penetrate it, are unable to migrate away through the agar.