According to Bacteria are simplest but complex and sophisticated organism as compare with other microorganisms. Bacteria are classified as prokaryote organisms due to lack of true nucleus and nucleus membrane, it chromosome lies freely in the cytoplasm. Morphologically, bacteria adopt variation of shape and size including irregular, circular, elevated, flat and punciform. However, bacteria are grouped in three main shapes namely rod -shaped (bacillus), spherical (coccus) and spiral (spirillus), these bacterial cell shapes are of fundamental significant in identification and classification of bacterialCITATION Ess13 l 7177 (Essays, 2013).
When classifying microorganisms, all known characteristics are taken into consideration, but certain characteristics are selected and used for the purpose of identification. Primary identification usually involves a few simple tests such as morphology (usually shown by Gram stain), growth in the presence or absence of air, growth on various types of culture media, catalase and oxidase tests1. Using these few simple tests it is usually possible to place organisms, provisionally, in one of the main groups of medical importance CITATION Due98 l 7177 (Duerden, Towner, & Magee, 1998).
Primarily, bacterial identification is the observation of some characteristic of unknown strain with registered bacteria strain or specie for example Escherichia coli. A number of specialised biochemical tests usually performed in bacteria identification includes carbohydrate test, enzyme test and test for specific end-products (API 20E kit). These tests often based on combination of morphology and metabolic by-product. Conversely, immunological tests, Protein and Nucleic acid sequences and typing method can also be used in obtaining specific and accurate conformation of true identities of strains of bacteria in a specialized controlled laboratory procedure. However, for the purpose of routine bacterial identifications, these techniques are very expensive and time consuming as compared to primary biochemical testCITATION Ess13 l 7177 (Essays, 2013).
Carbohydrates are complex chemical substrates, which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen and oxygen with hydrogen and oxygen in the same ratio as water and are usually classed as sugars CITATION Tor04 l 7177 (Tortora, Funke, & Case, 2004).
Facultative anaerobic and anaerobic bacteria are capable of fermentation, anaerobic process during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy. The types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism CITATION Rei13 l 7177 (Reiner, 2013).
We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced either acid end products, or acid and gas end products CITATION Rei13 l 7177 (Reiner, 2013).
In order to test for these fermentation products, inoculate and incubate tubes of media containing a single carbohydrate, a pH indicator (phenol red), and a Durham tube as it detect gas production. If the particular carbohydrate is fermented by the bacterium, acid end products will have produced, which lower the pH, causing the pH indicator to change colour (phenol red to yellow). If gas is produced along with the acid, it collects in the Durham tube as a gas bubble. If the carbohydrate is not fermented, no acid or gas will be produced and phenol red will remain red CITATION Tor04 l 7177 (Tortora, Funke, & Case, 2004).
Gelatin is a protein derive from the animal protein collagen, a component of connective tissue and tendons in human and other animals. It has been used as a solidifying agent in food for a long time. Robert Koch used nutrient gelatin as an early type of solid growth medium. Gelatin dissolves in water at 50? and exists as liquid above 25? and solidifies or gels when cooled below 25? CITATION MAc00 l 7177 (MAcfaddin, 2000).
Nutrient gelatin appears to be a liquid or broth medium when maintained at room or incubator temperatures, but will solidify when placed in an ice bath (or refrigerator) for a period of time (much like jello). When inoculated with microbial cultures and allowed to incubate for a period of time (24-72 hours), tubes of nutrient gelatin can readily show protein catabolism. If the gelatin is catabolized by the action of microbial enzymes, the medium will remain liquid when cooled (gelatin liquefaction has occurred and hydrolysis is positive). If the medium is solid when cooled, gelatin liquefaction has not occurred and hydrolysis is negative CITATION MAc00 l 7177 (MAcfaddin, 2000).
Triple sugar iron agar (TSI) is a differential medium that indicates the ability of organisms to ferment lactose, sucrose, and glucose, with the formation of acid and gas, and also their ability to produce hydrogen sulphide (H2S) within a single tube.
Inoculation of the medium with organisms able to ferment one or more of the carbohydrates present and produce acid will cause the phenol red indicator in the medium to change from red to yellow. This colour change may be visible throughout the slant or may be restricted to a certain area. In general, the results for glucose and sucrose fermentation are read in the tube butt (bottom) while the results for lactose fermentation are read in the slant (upper portion). Inoculation of TSI with Klebsiella pneumoniae or Klebsiella oxytoca (capable of fermenting all three sugars) will usually result in a colour change from red to yellow throughout the medium, while inoculation results in the production of acid and sometimes gas from the fermentation of sucrose and glucose, but not from lactose. This is indicated by a colour change in the butt of the tube, but no change in the colour of the slant. Organisms that can ferment glucose but form acetoin (a neutral end product) will often leave the slant portion of the TSI medium red (a false negative for lactose fermentation). Proteus vulgaris cannot ferment lactose, but will form enough acid through the fermentation of glucose and sucrose to turn the entire medium yellow (a false positive for lactose fermentation). Since the fermentation of glucose and sucrose are indicated together in the tube butt, and false readings for lactose fermentation are fairly common, TSI medium provides a less reliable indication of carbohydrate fermentation than do individual carbohydrate deeps.
The TSI medium also indicates the ability of organisms to degrade sulfur-containing amino acid molecules and produce hydrogen sulphide. The sulfur released as H2Swill bind with iron in the medium to form a black precipitate called iron sulphide (FeS). In some cases, H2S production is indicated by the formation of a small amount of black precipitate (iron sulphide) between the butt and the slant, in others the entire tube bottom will turn black. If iron sulphide turns the entire lower portion of the tube black, it will not be possible to read the results for sucrose and glucose fermentation.
Hydrogen sulphide (H2S) production test is used for the detection of hydrogen sulphide gas produced by an organism. It is used mainly to assist in identification organisms. H2S is produced by certain bacteria through reduction of sulphur containing amino acids like cysteine, methionine or through the reduction of inorganic sulphur compounds such as thiosulfates, sulfates or sulphites. The hydrogen sulphide production can be detected by incorporating a heavy metal salt containing iron or lead as H2S indicator to nutrient culture medium containing cysteine and sodium thiosulfates as the sulphur substrates. Hydrogen sulphide, a colourless gas, if produced reacts with the metal salt forming visible insoluble black precipitate of ferrous sulphide.
Urea is waste product excreted in urine by animals. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. The urease test is useful in identifying bacteria, which liberate this enzyme. Urease, which produced by some microorganisms, is an enzyme that is especially helpful in the identification of Proteus species. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. Two units of ammonia are formed with resulting alkalinity in the presence of the enzyme, and the increased pH is detected by a pH indicator. The presence of ammonia creates an alkaline environment that causes the phenol red to turn into deep pink. This is a positive reaction for the presence of urease. Failure of deep pink pink colour to develop is evidence of a negative reaction.
MATERIALS AND METHODS
Materials: 48 -24 hrs unknown broth culture 9.
A smear of the culture was prepared
Allowed to air dry and heat fixed.
The smear was flooded with crystal violet and allowed to stand for 1 minute.
Washed with tap water.
The smear was flooded with grams iodine and allow to stand for 1 minute.
Washed with tap water.
The smear was covered with 95% ethanol and allowed to decolourise for 20-30 seconds and washed with tap water immediately , alternately holding the slide at a slant a drop od 95% ethanol was added until crystal violet failed to washed from the smear.
The smear was flooded with safranin and allowed to stand for 45 seconds.
Washed with tap water and gently blot dryed with paper towel.
Examined under oil immersion.
Materials: broth cultures of an unknown culture 9.
: 2 nutrient agar plates
A loopful of the inoculum on the agar was placed on the surface and dragged lightly several times across the surface in straight line in area 1. The loop was touched at the end of series of lines in area 1 and draged several times across the agar in area 2 while ta agar was turned 90 0 making sure that the lines do not touch each other. In the same manner for area 3 it is done the same way and area 4 but in area 4 the streak lines are moved into the centre.
Incubated the cultures at 370 for 24-48 hours.
PRACTICAL 3: IDENTIFICATION OF GRAM NRGATIVE RODS 1
MATERIALS: unknown culture code 9
1x glucose broth (green),1x sucrose broth (dark blue),1x lactose broth (red),1x arabinose broth (black /orange), 1x inositol broth (yellow),1x manittol broth (purple),1x dulcitol broth (pink), 1x nutrient broth (sub culture unknown).
The two organism were inoculated into each of the carbohydrate broth, incubated at 37 degrees for 24 hours. The technician incubated each of the carbohydrate broth unioniculated that served as negative control.
MATERIALS: gelatin deeps
Gelatin deeps stab-innoculated into each of the two organism assigned to us. Incubated at 37 degrees for 7 days. The test tubes were removed from the incubator and held at 4 degrees for 30 minutes.
TSI AND HYDROGEN SULPHIDE PRODUCTION AND CARBOHYDRATE FERMENTATION.
MATERIALS: TSI agar with butt of about 2.5 cm
A stab inoculation was made to the organism into a butt of TSI slope and when the loop is withdrawn, zig-zag inoculated to the slope surface. Incubated at 37 degrees for 24 hours.
MATERIALS: SIM agar deep
The organism was stab inoculated into the SIM deep. Incubated for 24 to 48 hours at 37 degrees.
MATERIALS: Christiansen’s urea agar slopes
The two organism assigned to us were inoculated onto a slope with zig-zag inoculation. Incubated for 24 hours to 48 hours at 37 degrees.
Gram Stain and Streak plate
Table 1: describes gram reaction, shape and arrangement of the cell. Also streak plate describes the colonial morphology and appearance
Gram stain Pink-red, rods Gram-negative
Streak plate Colony was smooth, not dull but shiny, reflecting light and it was creamy white.
Margins: entire CARBOHYDRATE FERMENTATION
Results are recorded as follows:
AG: acid and gas
A: slightly acidity
O: carbohydrate not fermented
Table 2.1: summarises results obtained from carbohydrates fermentation after inoculating culture 9 (Klebsiella pneumonia)
CARBOHYDRATES ORIGNAL COLOUR COLOUR CHANGE
Glucose Orange-red Yellow (AG)
Sucrose Red Yellow (A)
Lactose Red Yellow (A)
Arabinose Red Yellow (A)
Mannitol Red Yellow (A)
Inositol Red Yellow (A)
Dulcitolred Red (O)
Table 2.2: summarises carbohydrates fermented by other unknown organisms
Organisms Glucose Sucrose lactose Arabinose Mannitol Inositol DulictolCulture 22 AG A A A A A O
Culture 13 AG AG A A A A O
Culture 18 AG A O A O O O
Table 3: describes results of Culture 9 (K. pneumonia) obtain from gelatin after incubation and refrigeration including other unknown organisms
Organism Gelatin result
Culture 9 (Klebsiella pneumonia) Negative, solid
Culture 22 Negative, solid
Culture 13 Negative, solid
Culture 18 Negative, solid
TSI-H2S PRODUCTION AND CARBOHYDRATE FERMENTATION
Table 4: describes how the Culture 9 (K. pneumonia) reacted in TSI-H2S production and carbohydrate fermentation
Species Culture 9 (K. pneumonia)
Butt Yellow butt
Slope Yellow slope
Cracks?/ gas production Cracks and gas production
Blackening?/H2S No H2S
Sugars utilised Yes
Table 5: shows how Culture 9 (K. pneumonia) responded to SIM agar
organism Culture 9 (K. pneumonia)
Colour of medium colourless
H2S production Negative
Table 6: describes how Culture 9 (K. pneumonia) possess the enzyme urease and hydrolyses urea.
Organism Culture 9 (K. pneumonia)
Colour of the medium Deep pink
The results obtained from the experiment indicate variation in growth as a result of favourable conditions provided for each organism throughout the incubation stage. Difference in bacteria colony appearance including shape, elevation, edge colour and texture were observed on the growing organism and this can be attributed to type of bacteria use in the experimentCITATION Ess13 l 7177 (Essays, 2013). Stated that bacteria thrive well when placed in an appropriate nutritious atmosphere maintained under right chemical and physical environments. This suggests that, the cell division leading to the multiplication of cell colonies on the growth medium was under suitable conditionCITATION Ess13 l 7177 (Essays, 2013).
Gram staining method reveals the cell morphology of various bacteria such as the rod, cocci. Gram staining can also assist in grouping bacteria into gram positive and gram negative using the cell wall composition. This confirms the observation discovered under the microscope, some bacteria exhibit the characteristics of gram positive and gram negative. The appearance of the colonies was grown from isolated bacteria on the surface of nutrient agar. The gram stained smear shows the gram reaction, shape, groupings of the bacteria as shown on table 1.
The term fermentation is often used to describe the braking down or catabolism of a carbohydrate under anaerobic conditions. Therefore, bacteria capable of fermenting a carbohydrate are usually facultative anaerobes. It should also be noted that while the terms carbohydrate and sugar are often used interchangeably, term sugar might not indicate the true chemical composition of certain substrates such as in the case of dulcitol CITATION Rei13 l 7177 (Reiner, 2013). The microorganism may produce acidic end product for example lactic acid and carbon dioxide, or neither acid nor gas from particular carbohydrate substrate. The product rely on the ability of the microorganism is skilled to carry out. Identification of the production of acid and gas can be carried out by using a medium CITATION Rei13 l 7177 (Reiner, 2013).
Normally, especially in aerobic respiration, the end products contain carbon dioxide. This is because oxygen is used as final electron acceptor. However, in anaerobic respiration, other inorganic substance like nitrate ion replace the role of oxygen as final electron acceptor CITATION Tor04 l 7177 (Tortora, Funke, ; Case, 2004). If nitrate ion is used, it will be reduced to nitrite ion, nitrous oxide or nitrogen gas. Certain microorganisms produce hydrogen sulphide as they use sulphate ion as final electron acceptor. Carbonate ion can also be used and the end product gas is methane. Therefore, not only carbon dioxide may found in the air bubble, other gases like nitrous oxide, nitrogen gas, hydrogen sulphide and methane gas could be found as well CITATION Tor04 l 7177 (Tortora, Funke, ; Case, 2004).
In the experiment, referring to table 2.1, there was visible change for all pH indicators and there was gas production only in glucose. Except for dulcitol, no carbohydrate was fermented nor gas production. This may due to the culture 9 (Klebsiella pneumonia) fail to grow well on the broth. As there is no carbohydrate is used for cellular respiration, acidic end products are not produced. In table 2.2, other unknown organisms fermented the same as culture 9 (Klebsiella pneumonia). Except for culture 18, in which lactose, mannitol, inositol and dulcitol was not able to grow in broth leading to no acidic end product were produced.
This gelatin test is used to determine the ability of an organism to produce extracellular proteolytic enzymes, gelatinases that hydrolyse gelatin. The reaction occurs in two sequential steps; in first reation gelatinases hydrolyse gelatin into polypeptides and then polypeptides are further converted into amino acids. The presence of gelatinase is detected using a nutrient gelatin medium. This medium is a simple medium composed of gelatin, peptone and beef extract. When nutrient gelatin tube is stab-inoculated with a gelatinase positive organisms, the secreted gelatinases will liquefy the gelatin, resulting in the liquefaction of the medium. While the gelatinase negative organisms do not secrete enzymes and do not liquefy the medium but it solidifies CITATION MAc00 l 7177 (MAcfaddin, 2000).
Gelatin is an incomplete protein, lacking many amino acids, such as tryptophan. When collagen is heated and hydrolysed, denatured protein gelatin is obtained. Collagen accounts for 90% to 95% of organic matter in the cell. It is the most important protein, rich in amino acids. Microorganisms like bacteria can use gelatin only if they are supplemented with other protein. Bacteria produce the gelatin hydrolysing enzyme gelatinase CITATION Bio18 l 7177 (Biocyclopedia, 2018). Since gelatine is good solidifying agent at low temperature, its property of solidification can be used to distinguish between gelatin hydrolysing and non-hydrolysing agents. Most of the Enterobacteriaceae are gelatin hydrolysis test are negative CITATION Bio18 l 7177 (Biocyclopedia, 2018), including culture 9 (Klebsiella pneumonia) and other unknown cultures that were collected from other students, those results are shown above in table 3.
TSI-H2S PRODUCTION AND CARBOHYDRATE FERMENTATION
Lactose and sucrose fermentation has occurred. Since these substances are present in higher concentrations, they serve as substrates for continued fermentative activities, both carbohydrates were utilised, a lot of acid is produced and the medium turned yellow and results in gas production. The gas production was recognized within the agar itself, gas production cracked the agar. There was no H2S production, which means culture 9 (Klebsiella pneumonia) did not utilize thiosulfate anion. And no black precipitate was formed in the medium. The medium is designed to detect the Enterobacteriaceae, and because it contains no selective agents it will support the growth of a wide range of bacteria. The mere presence of the growth on the medium, therefore has no significance. It is the characteristics of the growth that is important CITATION vla11 l 7177 (vlab.amrita.edu, 2011).
Some bacteria are capable of breaking down sulphur containing amino acids (cysteine and methionine) or reducing inorganic sulphur-containing compound (such as sulphite, sulphate or thiosulfate) to produce hydrogen sulphide (H2S). This reduced sulphur may then be incorporated into other cellular amino acids or perhaps into coenzymes. The ability of an organism to reduce sulphur-containing compounds to hydrogen sulphide can be another test for identifying unknown organisms. To test for hydrogen sulphide production, a medium with a sulphur-containing compound and iron salts reduced and if the sulphur is reduced and hydrogen sulphide is produced, it will combine with with the iron salt to form a visible black ferric sulphide in the tube CITATION Cla53 l 7177 (Clarke, 1953).
SIM (Sulfur Indole Motiliy) is a differential medium that can be used to demonstrate hydrogen sulphide production and indole production in a single tube. The medium also contains material that allows for the determination of motility. Non-motile organisms will grow only along the line of inoculation (the line will be distinct and easily visible), while motile forms will show diffuse growth or turbidity throughout the medium CITATION vla11 l 7177 (vlab.amrita.edu, 2011). Table 5 describe culture 9 (Klebsiella pneumonia), there was line of inoculation which means K. pneumonia is a non-motile organism, no H2S production and the medium was colourless.
Urea agar was formulated to differentiate rapid urease positive organisms from slower urease-positive and urease negative bacteria. It contains urea, peptone, potassium phosphate, glucose, and phenol red. Peptone and glucose provide essential nutrients for a broad range of bacteria. Potassium phosphate is a mild buffer used to resist alkalinisation of the medium from peptone metabolism. Phenol red, which is yellow or orange below pH 8.4 and red or pink above, is included as an indicator. Urea hydrolysis (in table 6) to ammonia by urease positive organism that overcome the buffer in the medium and change it to pink. Rapid urease-positive organism turns the entire slant pink within 24-48 hours. Urease negative organisms either produce no colour change in the medium or turn it yellow from acid productsCITATION Pub15 l 7177 (England, 2015). Urea broth differs from urea agar in two important ways. First, its only nutrient source is a trace (0.0001 %) of yeast extract. Second, it contains buffers strong enough to inhibit alkalinisation of the medium by all but the rapid urease-positive organisms mentioned above. Other organisms, even some that would ordinarily be able to metabolize urea, cannot survive the severe nutrient limitations or overcome the stronger buffers in urea broth. Pink colour in the medium in less than 24 hours indicates a rapid urease positive organismCITATION Pub15 l 7177 (England, 2015).
Christensen’s urea medium contains the pH indicator phenol red which under acid conditions (pH 6.8) is yellow. In alkaline conditions (pH 8.4) the indicator turns the medium rose pink. Urea is unstable and broken down at psi or pressure. It cannot be added to the medium for autoclaving and is therefore filter sterilized and added to the medium after autoclaving CITATION Pub15 l 7177 (England, 2015)
Based on information conducted above, the K. pneumonia, the unknown organism (culture 9). Identity was reached after a series of tests and subcultures were conducted using aseptic techniques. Aseptic techniques are necessary in order to avoid contamination of the culture being examined
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Reiner, K. (2013). Carbohydrate fermentation. Microbe Library. Retrieved September 26, 2018, from http://microbelibrary.com/library/laboratory/laboratory-test/3779-carbohydrate-fermentation-protocol
Tortora, G. J., Funke, B. R., ; Case, C. L. (2004). Microbiology: an introduction. New York: Pearson Education, Inc.
vlab.amrita.edu. (2011). Triple Sugar Iron Agar. Retrieved September 30, 2018, from http://vlab.amrita.edu/?sub=3;brch=76;sim=216;cnt=2