Analytical microbiology lab report Name : Apu sarwar Class : analytical and pharmaceutical chemistry Date of practical: 2/10/12

and 9/10/12 and 16/10/12

Title: Estimation of the prevalence of nasal carriage of Staphylococcus aureus in the nasal passages of this class of college students.(three part all together) Objective: This prospective investigation on healthy college student is undertaking to estimate the prevalence of S.aureus nasal carriage and identify the micro organism by using different media and test like gram stain. Introduction: Staphylococci are often found in the human nasal cavity (and on other mucous membranes) as well as on the skin. S. aureus is with some frequency found as normal human flora, Approximately 30% of adults and most children are healthy periodic nasopharyngeal carriers of S. aureus. Staphylococcus aureus (coagulate-positive staphylococci). Staphylococcus aureus is the most pathogenic species and is implicated in a variety of infections. to diagnostic them it is very important to identify this microorganism. there is many way the micro organism can identify. Here different media and staining will apply to identify the organism. Method: for material and method the lab manual was followed. Result : Table 1 : Identification of organisms from the nasal swab in class 1. Identification technique Gram stain appearance Mixed(purple and pink) Results Gram positive and gram negative cocci are present

Table 2: swab from nose to msa and nutrient agar class 2. no 1 2 name of agar MSA nutrient agar colour of the colony Yellow colour less colony AGI 2 1

Table 3: organism from MSA and nutrient agar to DNA agar. (Analysis before put HCl) no 1 2 name of agar MSA nutrieant colour of the colony whites NA AGI 2 1

Table 4: Analysis after put HCl. chemical added HCl waiting time 10 minute change in the colony Clear zoon around the colony results Positive

Table 5: Total result in class S. aureus Number of student posotive 34 25

S.aureus negetive 9

% % positive negative 74 26

Discussion: Nasal swabs were obtained with cotton-wool swabs. The left and right anterior nares were swabbed four times around. The swabs were immediately inoculated in to two different medium Mannitol salt agar (MSA) and in a Nutrient agar. The Mannitol salt agar (MSA) was incubated at 37C (7 days); the Nutrient agar culture plates were also incubated at 37C (7 days). Identification of S. aureus was based upon colony morphology and gram stain. Gram stain was performed for the nasal sample during the 1st class for this practical. A mix result was found in the microscope from the stain. There was some pink and purple bacteria ware present. which means the gram positive and gram negative coocoi was present in the sample because gram negative bacteria lost their cell wall by stain and get pink in colour and gram positive stay purple. the result was mixed because there other bacteria could have been present in the nose sample or it could have been contaminated from air.but in the experiment the target bacteria is S.aureus. The AGI was counted by analysis of the growth of the bacteria on the agar in class 2 and it was 2 for MSA and 1 for Nutrient agar and the colony was yellow on the MSA agar. on the other hand the colony on the nutrient agar was colour less because it is not selective media to identify the bacteria it grow all the bacteria equally. But the MSA was yellow because the bacteria ferment mannitol, as a result the agar was changed the colour of the medium from red to yellow. Which means the Staphylococci ware present in the sample. So to identify the bacteria more specifically further identification technique was used. DNAse agar was used to do the confirmation. sample from Nutrient agar and the MSA agar was taken and inoculated onto DNAse agar with a control And incubated at 37C. (on class 2.) The DNAse agar was used because this agar is used to indentify the pathogenic staphylococci which are the target organism for this practical. In the 3rd day of the class the dnase agar was analysis and it was observed that all the sample was grown. to identify the pathogen bacteria, the agar was flooded with 1N HCl for 10 min and then the agar was analysed again there was a clear zone around the colony. which indicated that the pathogenic staphylococci was present.

Because at the present of HCl the DNA is polymerised and medium gets opaque, and the organism produce DNAse enzyme and hydrolysis the DNA and make clear zoon around the colony. In the sample there was Staphylococcus aureus because the clear zone was in large area which indicate that it was Staphylococcus aureus not Staphylococcus epidermidis because epidermidis produce very small clear zoon. There was 34 student was given the sample from there nose and 74 % was found S aureus positive. which was higher then expect value. The reason could be the some human error during the experiment also there could have some problem with identification of the clear zoon in the DNAse agar. Conclusion: This study provides a representative assessment of carriage of S aureus in the college student.The results suggest that nearly one in four of the population is currently not colonized by S aureus.