Screening Of Bacteria Isolated From Earthworm Cast For Antibacterial Activities

MATERIALS AND METHODS

1. 1. STUDY AREA

The study area was in Nsukka, a local government area in Enugu state, situated in Nigeria. The temperature range was about 23-30oc. samples were taken from the environment of the university of Nigeria, Nsukka and Odenigbo street in Nsukka.

1. 1. COLLECTION OF SAMPLES

Two relative samples were collected during rainy season in the month of June and July from different locations; on the road paths of the university of Nigeria and along the hills of Odenigbo in Nsukka, Enugu. The earthworm castings were carefully taken with spatula and kept in a jar. The collected earthworm castings were taken to the laboratory for isolation.

1. 1. MEASUREMENT OF pH

The pH of the samples was determined with a pH meter. Ten grams of each samples was suspended in 20mL of distilled water and allowed to stand for 20minutes with occasionally stirring to reach equilibrium. After being left to settle, the pH was measured.

1. 1. MATERIALS AND EQIUPMENT

Some of the major materials include; pipettes, glass wares (test tubes, petri dishes, conical flasks, beaker, glass rod, bijou bottles etc.), wire loop, gloves, cotton wool, masking tape, swab sticks, foil, syringes, antibiotics sensitivity disks, distilled water.

Equipment used include; pH meter, incubator, autoclave, weighing balance, refrigerator, microscope, gas cylinder, Bunsen burner.

1. 1. STERILIZATION OF MATERIALS

All glass wares such as test tubes, conical flasks, beaker etc. were sterilized in an autoclave at 121oc for 15 minutes. Prepared media was also sterilized in the autoclave at 121oc for 15 minutes. The wire loop was sterilized over a Bunsen flames.

1. 1. PREPARATION OF MEDIA/AGAR NUTRIENT AGAR

Nutrient agar is a general purpose, nutrient medium used for the cultivation of microbes supporting growth of a wide range of non-fastidious organisms. It is composed of 0.5% peptone, 0.3% beef extract, 1.5% agar, 0.5% NaCl, and distilled water. pH is adjusted to neutral (7.4) at 25oc. it is prepared according to manufacturer’s instruction (23g of ready-made agar in 1000ml distilled water).

The mixture is dissolved in distilled water, then heated to fully dissolve all components, after complete dissolving the mixture is put in the autoclave at 121oc for 15 minutes. The media is allowed to cool after autoclaving but not to solidify, the media is poured into petri dishes for use and allowed to gel in plates, finally the media is sealed and stored in a refrigerator.

MACCONKEY AGAR

MacConkey agar contains crystal violet and bile salts to prevent the growth of Gram-positive bacteria and fastidious Gram-negative bacteria. It’s an indicator, a selective and differential culture medium for bacteria designed to selectively isolate gram-negative and enteric bacilli and

differentiate them based on lactose fermentation. The media consist of peptone, polypeptone, lactose, bile salts, sodium chloride, neutral red, crystal violet and distilled water.

This agar was prepared by; suspending 49.53g of ready-made agar in 1000ml distilled water, then heat to completely dissolve powder, autoclave at 121oc for 15 minutes then allow to cool and use then refrigerate.

MUELLER HINTON AGAR

Nowadays, it is more commonly used for the susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique. MHA is composed of beef extract, acid hydrolysate of casein, starch, agar and distilled water. The suitable pH is 7.4 at 25oc.

The Mueller Hinton agar was prepared by dissolving 38grams of the ready-made agar in 1000ml of distilled water, it was heated and then autoclaved at 121oc for 15 minutes. After preparation, the agar was stored at 25oc and pH of 7.4.

STARCH AGAR

Starch agar is a differential medium that tests the ability of an organism to produce the extracellular enzymes a-amylase and oligo-1,6-glucosidase that are secreted out of the bacteria and diffuse into the starch agar. These enzymes hydrolyze starch by breaking the glycosidic linkages between glucose subunits and allow the products of starch hydrolysis to enter the cell. Starch agar is also used to differentiate members of various genera which have both amylase-negative and amylase- positive species. Starch agar is composed of beef extract, soluble starch, agar and distilled water.

This agar was prepared by completely dissolving ready made starch agar powder in distill water through heating (excessive heating was avoided to prevent hydrolyzing the starch) then autoclaving for 15 minutes at 121oc. The agar was poured into petri dishes and allowed to solidify before use.

1. 1. ISOLATION OF BACTERIA

The samples taken from the field were dissolved in 10mL sterile normal saline and serially diluted up to 10-7 and plated (0.1mL) on nutrient agar and MacConkey agar. The serial dilution was done by pouring 9mL of water into seven test tubes then adding 1mL of the dissolved samples into the first test tube (10-1) and then adding 1mL into the rest of the test tubes from the previous test tube of each.

After serial dilution, the 10-5, 10-6, and 10-7 dilutions of the samples were plated on both nutrient and MacConkey agar. The pour plate method was used and 0.1mL of each dilution was poured and spread in petri dishes already containing the mentioned agar. The petri dishes were transferred to the incubator where the organisms were cultured (about 20-24 hours).

Sub culturing was done after there was appearance of growth in the cultured plates. Different colonies were picked from each plate (using a sterilized wire loop) and inoculated into separate plates containing nutrient agar. After about 24 hours the sub cultured inoculates were isolated into slants in bijou bottles and kept as stock or isolates.

1. 1. IDENTIFICATION OF BACTERIA

The morphology of the isolates was characterized by their size, colour, shape, edges etc. They were gram stained and then viewed under the microscope (100x lens was used).

1. 1. 1. GRAM REACTION

Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups (Gram-positive and Gram-negative).

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out. They are stained pink by the counterstain,[2] commonly safranin or fuchsine. Gram’s reagent is prepared by applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, adding iodide which binds to crystal violet and traps it in the cell then rapid decolorization with ethanol or acetone. After decolorization counterstaining with safranin is done.

1. 1. 1. BIOCHEMICAL CHARACTERIZATION

Biochemical test was also carried out on each isolate. A few biochemical tests were used to identify the isolates, the tests carried out includes;

CATALASE TEST

Tests for the presence of catalase, an enzyme that breaks down the harmful substance hydrogen peroxide into water and oxygen. If an organism can produce catalase, it will produce bubbles of oxygen when hydrogen peroxide (H2O2) is added, this interprets for a positive result.

3% H2O2 was placed (a drop) on different sterile glass slides and the test colonies were smeared on the reagent. The results were then interpreted.

OXIDASE TEST

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine) to purple (indophenols) colour end product which interprets a positive result. A colourless end product interprets a negative result.

The filter paper was soaked with few drops of the substrate tetramethyl-p-phenylenediamine dihydrochloride. The paper was moistened with a sterile distilled water. The colony to be tested with was picked and smeared with a sterile wire loop in the filter paper then the results were noted.

STARCH HYDROLYSIS TEST

The major component of starch can be hydrolyzed by a-amylase, which is present in some bacteria hence the ability to degrade starch is used as a criterion for the determination of amylase production by a microbe, and also determine the specie of bacteria.

A sterile technique was used to make single streak inoculation of the isolates into the Centre of labeled plate. The bacterial inoculated plates were incubated for 48 hours at 37oc. following incubation, the surface of the plates was flooded with iodine solution with a dropper for 30 seconds. The excess iodine was poured off and the result was read. A clear zone around the line of growth after addition of iodine solution indicates that the organism has hydrolyzed starch (positive result). A total blue -black coloration of the plate interprets a negative result.

INDOLE TEST

This is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. Pure culture of the isolates was grown in peptone broth for 24- 48 hours. Following incubation, five drops of kovac’s reagent was added to culture broth. A positive result is shown by the presence of a red or red-violet colour in the surface layer of the broth. A negative result appears yellow.

1. 1. SCREENING OF BACTERIA

The bacterial isolates were screened for antibacterial activity using the agar plug method. The isolates were spread on nutrient media and as soon as the isolates grew, agar discs were cut out (specifically around the isolate axis) with the cork borer. These discs were transferred to the surface of Mueller Hinton agar plates already seeded with the test organisms. Initially, the test organisms were spread separately on to Mueller Hinton agar plates using sterile glass rod. The petri dishes were then kept in the incubator at 37oc for about 24 hours. If there is a clear zone around the disk, the isolate produces antibiotics that inhibits the growth of the test organism.

The test bacterial organisms were pure clinical isolates obtained from the Diagnostic Microbiology laboratory, Departments of Microbiology, University of Nigeria, Nsukka. These include one isolate of some of the human pathogens: Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli