Remove excess water from the surface of the gram stained glass slide and observed under 1000X oil immersion microscope. Gram-staining have performed for Staphylococcus aureus (control); Enterococcus faecalis (control); Nostril microflora in NA, MSA, and PYCa. Gram-staining provides results of the bacteria morphology, type of gram-stain. Catalase test was also done prior to gram-staining. MicrobactTM Biochemical Identification Kit was used for identifying gram-negative aerobic and facultatively anaerobic bacteria.
Gram Stain Test The Gram Stain test was first performed to differentiate the bacteria based on the thickness of the peptidoglycan layer in its cell wall. The unknown bacteria appeared purple and round, therefore indicating a gram-positive cocci bacterium. The purple color was observed because S. agalactiae had a thicker layer of peptidoglycan in its cell wall, therefore it allowed for the cell wall to not be stripped away after the addition of ethyl alcohol, but rather enabled for the peptidoglycan to form pores (Madani, 2003). The pores then trapped the crystal violet-iodine complex, which produced the purple color of the bacteria cells that were observed under the light microscope. Catalase Test To further identify the gram positive unknown bacteria, the Catalase test was conducted in order to differentiate the gram-positive species into the Staphylococcus species (catalase-producers) or the Streptococcus species (catalase
Three bacterial cultures were received from laboratory staff. The colonies that cultures were grown from came from ligations and transformations that were prepared by the laboratory staff. The cultures prepared during the ligation and transformation steps were unusable due to unsuccessful ligation. Two colonies were taken from an LB/kanamycin/IPTG plate. One that fluoresced green under UV light, and one that did not fluoresce green under UV light.
al.. Cyclodextrin glucosyltransferase (CGTase) producing alkalophilic bacteria were isolated from the water samples collected from Marneri pond of Tirunelveli, Tamil Nadu by serial dilution and plating method. Totally 22 bacteria were isolated from the collected water samples and screened for CGTase activity by Horikoshis medium II agar plate method. Among 22 bacterial isolates, 15 isolates showed CGTase activity and better zone producing strain was selected for further studies. The potential strain was identified as Bacillus circulans by 16S rDNA gene sequencing experiment. The best enzyme activity was observed at pH 10.5, 32°C, supplemented with cassava as carbon source an d beef extract as nitrogen source.
Since the stomach is highly acidic by a PH range of 1 to 2 the listeria survives that acidic medium and it is passed down the small intestines with the food bolus then When it is in the intestines it targets different areas of the intestinal epithelium such as payer’s patches and intestinal villi. It targets the tip of the villi where apoptotic cells are removed or the lateral goblet cells specializing in mucus secretion. The crossing of intestinal barrier starts with the interaction of the listeria protein called internalin-A with E-cadherin which is a specific receptor of the cell. Afterward, the bacteria then enter the goblet cell to the lamona propea to the bloodstream it then secrets lysine-O toxin that makes pores on the phospholipid membrane through which ions pass in and out leading to ion imbalance of the cell which then promotes bacterial entrance by zipper mechanism by compromising cell internal processes and organelles. The excess calcium entering the cell causes ionic imbalance leading to (1) mitochondrial fragmentation (2) histone dephosphorylating (3) transcription complications and ion desumoylation.
The swab was inoculated onto blood agar, MacConkey’s agar and nutrient agar, mannitol salt agar and Sabouraud's dextrose agar and was incubated at 370C for 24 hrs for bacterial culture and at room temperature for fungal isolates. Next day the colonies were picked up and preliminary identification was done and the bacterial isolates were identified based on standard protocols. LPCB was done for the identification of fungal isolates after 48 to 72 hrs. ANTIBIOTIC SUSCEPTABILITY TEST Antibiotic sensitivity test of the bacterial isolates was determined by the Kirby-Bauer disc diffusion method2. The following were the antibiotics used for the study-amikacin (AK 30), nalidic acid (NA 30), erythromycin (E 15), vancomycin (VA 30), tetracycline (TE 30), cefoxitin (CX 30), rifampicin (RIF 5) ciproflaxicine (Cip 5), ceftazidime (CAZ 30), cefotaxime (CTX 30), cepifime (Cpm 30), cefoperazone (CPZ 75).
Tube 2 under UV light after addition of wash buffer. Figure 2. Tube 3 under UV light after addition of elution buffer. Before doing chromatography, lysozyme was first added in order to break open the cell wall and release the components of the cell then it was centrifuged then binding buffer was added to the supernatant. The binding buffer allows the GFP to change its confirmation to expose its’ hydrophobic particles because of the presence of high amount of salt.
Of all the kefir starter microbial components, the microphillic homofermentative lactococci and acetic acid bacteria are the most active against coliforms.Van Wyk (2001) showed that kefir possesses an inhibitory activity against Staphylococcus aureus,Bacillus cereus,Escherichia coli,Clostridium tyrobutyricum and Listeriamonocytogenes.Studies have also indicated that yeasts such as Torulaspora, whenseparated fromkefir, possess pronounced antimicrobial activity against coliforms (Powell, 2006 and Garrote,et al., 2010).The exact cause of the inhibition is not known, but may be due to the antagonistic action of various species of lactic acid bacteria (LAB) (Garrote,et al., 2010 and Magalhães,et al., 2011). Lactic acid bacteria are also capable of preventing the adherence, establishment, replication, and pathogenic action of certain enteropathogens (Rea, et al. 1996).The
The procedure of staining will distinguishes organisms of the domain bacteria according to structure of cell wall. A thick peptidoglycan layer and stain blue to purple indicate as the Gram-positive cells while a thin peptidoglycan layer and stain red to pink indicate as Gram-negative cells. In bacteriology, the most frequently used staining procedure is the Gram stain, is a complex and differential staining procedure. Organisms in the Domain Bacteria are differentiated according to cell wall composition over and done with a series of staining and decolorization steps. Cell walls of Gram-positive bacteria contain thick layers of peptidoglycan which 90% of cell wall and this stain are purple.
Lactobacillus lactis was inoculated in various media containing different vegetal source instead of beef extracts and was compared to the MRS culture medium as standard. These were incubated at 37oC for 72 hours wherein the growth was recorded every 24 hours using an undiluted, 10-4, 10-5 and 10-7 CFU/ml. The results of cultivation showed that the alternative test media using seed powders demonstrated better growth of colonies than the Man Rogosa Sharpe (MRS) cultre medium. Orange juice was used as an enrichment means for the enumeration of Alicyclobacillus acidoterrestis. In this process the inoculum was suspended in a medium containing orange juice and inoculated to different media such as K Agar, Yeast Starch Glucose Agar, Alicyclobacillus Agar, and Bacillus acidoterrestis Thermophilic Agar using duplicates for each run (Anjos et al, 2014).