Title: culture identification through culture characteristics, appearance, grams staining reactions
and catalase tests of cultures K & J.
Aim: To identify the bacteria in culture K & J through culture appearance, staining
characteristics and microscopic examination by the gram staining method.
A fundamental method of studying bacteria is by culturing them in liquid media or on the surface
of media that have been solidified by agar. Media contain nutrients, varying from simple sugars
to complex substances such as meat broth. To purify or isolate a single bacterial species from a
mixture of different bacteria, solidified media generally are used. Individual cells dividing on the
surface of solidified media do not move away from each other as they do in liquid, and after
many rounds of replication they form visible colonies composed of tens of millions of cells all
derived by binary fission from a single cell. If a portion of a colony is then transferred to a liquid
medium, it will grow as a pure culture free of all other bacteria except the single species that was
found in the colony.
Many different species of bacteria so closely resemble one another in appearance that they
cannot be differentiated from one another under the microscope. Various culture techniques have
been developed to aid in species identification. Some media contain substances to inhibit the
growth of many bacteria, but not the species of interest. Others contain sugars that some but not
all bacteria can utilize for growth. Some media contain pH indicators that change color to
indicate that a constituent of the media has been fermented, yielding acid end products. Gas
production as an end product of fermentation can be detected by inoculating bacteria in solidified
media in tubes rather than on plates. Sufficient gas production will result in the formation in the
agar of bubbles that can easily be seen. Still other media are formulated to identify bacteria that
produce certain enzymes that can break down constituents in the media; for example, blood agar
plates, which can detect whether bacteria produce an enzyme to lyse, that is, dissolve, red blood
cells. The various culture media and culture techniques are essential to the hospital laboratory,
whose job it is to identify the cause of various infectious diseases.Sterilization is maintained
during this experiment: Drying or freezing kills many species of bacteria and causes others to
become inactive. Heat or moist heat above a certain temperature kills all bacteria. Sterilization of
many different objects, such as spacecraft and surgical instruments, are important facets of
There are several number of bacteria that is gram positive and these include;
STAPHYLOCOCCUS, STREPTOCOCCUS, CORYNRBACTERIUM, LISTERIA, ERYSIPELOTHRIX, CLOSTRIDIUM,
BACILLUS, ACTINOMYCES, NOCARDIA, MYCOBACTERIUM and DERMATOPHILUS
Culture appearances refer to the physical or grossly viewed features such as colonies on the
plate: size, shape, elevation, texture and colonies. Hemolysis in blood agar and lactose
fermentation in MacConkey agar were also taken note of by simple gross observation. Besides
being differential, MacConkey is used to detect bacteria that produces hemolysin, a substance
that is capable of lysing red blood cells. The lysis appears around the colony on the agar plate
and it is used to identify the characteristics.
Streptococcus exhibits a kind of hemolysis called alpha hemolysis, characterized by a greenish
zone around the colonies (Nester, E, L et al, 2004).
Some bacteria are covered with a layer of firm gelatinous material in direct contact with the cell
wall and visible by light microscopy. This material is the capsule. The capsule is normally
composed of complex polysaccharides. Streptococcus pneumoniae (Pneumococcus) is an
organism with prominent capsule composed of polysaccharide. Capsules may also take the form
of a very thin microcapsule or a loose layer of slime. The form of capsule is influenced by the
culture conditions. The bacterial genera streptococcus and staphylococcus belong to the family
Enterobacteriaciae together with several other bacteria such as Escherichia, Salmonella,
Brucellaceae, Brucella, Bacteroidaceae, Sphaerophorus, Micrococcaceae, Lactobacillaceae,
Corynebacteriaceae and Corynebacterium.
The structure of the cell wall in bacterial are more complex than those of eukaryotic cells and
they contain substances unique to bacteria. Peptidoglycan, formally called mucopeptide is the
most important component of the bacterial cell wall; being common to both Gram positive and
Gram-negative cells. It surrounds the cell external to the cytoplasmic membrane as a single baglike molecule. It is composed of linear glycan chains of alternating residues of N-acetyl
glucosamine and N-acetyl muramic acid. These are linked together by short peptide bridges to
form a cross-linked insoluble polymer. Thus, peptidoglycan forms the basic structure of the
bacterial cell wall.
In addition to peptidoglycan, other accessory polymers are also found in most bacteria. Gram positive organisms such as staphylococci, contain teichoic acids composed of either polyglycerol
phosphate or polyribitol phosphate. These occur both within and on the surface of the cell wall
and may account for 20-50% of the cell wall mass.
In Gram - negative cells, the peptidoglycan is much thinner than in Gram - positive organisms.
External to the peptidoglycan lies a membrane (the outer membrane) which contains different
proteins and a unique component, lipopolysaccharide (endotoxin). Endotoxin is important in
pathogenicity of some disease and is also known as somatic O antigen. The LPS is responsible
for many of the biologic activities associated with gram negative bacteria. A major protein of
outer membrane is called porin which forms transmembrane pores or diffusion channels.
Teichoic acid / Teichuronic acid
Fig1. Principal components of cell wall of gram positive and gram negative components.
Gram’s stain is the most widely used differential stain. Gram-positive bacteria stain purple and
gram-negative bacteria appear pink. The primary stains (crystal violet and iodine) enter the
bacterial cell and, brief treatment with a decolourising agent such as acetone or alcohol removes
the stain from Gram-negative bacteria but not from intact Gram-positive bacteria. The
decolourised bacteria are then counter stained with a simple stain (carbol fuchsin) which allows
the Gram-negative organism to be seen. The time allowed for decolourisation is critical as too
little will leave Gram-negative organisms apparently positive and too much will over decolourise
Gram-positive organisms so that they appear to be negative. The uses of the Gram’s stain reflects
basic differences in cell-wall structure the reaction of bacteria to it is widely used in
classification and indicates how an individual species may react to antibiotics such as penicillin.
Gram positive bacteria are more susceptible than Gram-negative bacteria to the antibacterial
actions of penicillin, acids, iodine, basic dyes, detergents and Lysozyme and less susceptible to
alkalines, azide, tellurite proteolytic enzymes and lysis by antibody and complement. The
mechanism of the Gram stain is not fully understood. One factor of importance is that the
protoplasm of the Gram-positive bacteria is more acidic. Another is that the cell wall of Gram
positive bacterium is less permeable
Microscopic examination; the microscope is one of the most important tools used in studying
bacteria. Dyeing or staining bacterial specimens or cultures was introduced in 1871 by the
German pathologist Karl Weigert and has greatly helped the bacteriologist in identifying and
observing bacteria under the microscope. A bacterial specimen is first placed on a glass slide.
After the specimen has dried, it is stained to render the organism easier to observe. Stains also
stimulate reactions in certain bacteria. For example, the tuberculosis bacillus can be recognized
only on the basis of its reaction to certain stains. Bacteriologists have been greatly aided by the
electron microscope, which has far greater magnification powers than ordinary microscopes. The
gram stain is a differential stain method that distinguishes between gram positive and gram
negative bacteria. Differences in the cell wall composition of gram +/- bacteria accounts for the
characteristic stain. (Encarta, Microsoft student, 2009)
Prepared cultures of K & J on blood agar and MacConkey
Microscopic glass slide
crystal violet dye
Lugols iodine solution
Slide preparation & fixing
The slide was cleaned in order to remove all traces of grease
A loop full of normal saline or distilled water sterile was transferred onto the glass slide
Using a sterile loop, the surface of an independent colony was touched gently
The loop full was emulsified/mixed with a drop of normal saline and it was spread
The slide was then left to dry on the bench at room temperature
The slide was then fixed over a Bunsen burner flame by passing it over the flame 3 times
–noting that the surface with bacteria should be on top so as to kill the bacterial thereby
making it less viable and also to make bacteria to adhere to the slide.
7. The slide was left to cool and the staining procedure followed.
Staining: Gram Stain procedure
1. The slide was flooded with crystal violet solution for 1 minute
2. The stain was washed off under running tap water
3. The slide was Counter stained with lugols iodine; a mordant that does not take part in the
actual staining but only enhances the performance of crystal violet
4. The slide was Washed off again under running tap water
5. The slide was decolorized using 70% alcohol /acetone in brief
6. The slide was washed off under running tap water
7. The slide was counter stained with dilute safranin/ carbofushin
8. The slide was washed off under running tap water
9. The slide was then left to dry
10. An oil drop was added on the slide to enhance visibility under the light microscope
11. The slide was examined under a light microscope at x100 oil power
Fig2: showing results of different physical/microscopic bacterial parameters
No growth in
In this laboratory practical several methods of culture identification were employed and it was
observed that some evidence streptococcus and staphylococcus bacteria were noted. On blood
agar plate the observations that were made were the same for culture K & J: entire shaped,
positive hemolysis and greyish in color. The exceptions were in terms ofsize: where k was 0.51.0mm and J was 2mm. Another difference is in the arrangement of the cocci: k was long
chained, while J was clustered like grapes. Most bacteria come in one of three shapes: rod,
sphere, or spiral. Rod-shaped bacteria are called bacilli. Spherical bacteria are called cocci, and
spiral or corkscrew-shaped bacteria are called spirilla. Some bacteria come in more complex
shapes. A hairlike form of spiral bacteria is called spirochete. Streptococci and staphylococci are
well-known disease-causing bacteria among the cocci.
Streptococcus, genus of spherical, gram-positive, aerobic bacteria. The streptococci occur in
pairs or chains, and some species are pathogenic in humans. Streptococcal infections include
strep throat, scarlet fever, erysipelas, puerperal fever, and some pneumonias. The drugs of choice
for treating such infections are penicillin and erythromycin. Cultures of nonpathogenic lactic
streptococci are used in the fermentation of dairy products such as cheese and buttermilk
(Microsoft ® Encarta ® 2009).
Staphylococcus, genus of round, parasitic bacteria, commonly found in air and water and on the
skin and upper part of the human pharynx. These bacteria are known to cause pneumonia and
septicemia as well as boils and kidney and wound infections.