Microbiology Lab Report

MICROBIOLOGY LAB REPORT INTRODUCTION Bacterial identification has significant importance in many different industries including the pharmaceutical industry, when considering microbial contamination. Microbial contamination refers to the accidental introduction of an infectious material, such as bacteria, yeast, fungi or virus, to the production line (Donaldson, 2003). By inspecting for microbial contamination in products, it ensures quality control as there won’t be any damage, namely in the way of chemical degradation, which can lead to the loss of efficacy as well as toxic side effects and medicine spoilage, all of which can be detrimental to a patient’s health. There would also be a great loss in profit therefore it is an essential investment for a pharmaceutical company to take. Analytical techniques, for example, morphological, biochemical and genetic sequencing tests, can be performed in order to identify different types of microorganisms. By identifying bacteria, sterilisation of products can take place in the hope of removing pathogens and reducing the risk of contamination. Contamination can occur at any stage of production; however companies regularly assess the purity of a product on a frequently timed schedule to ensure an infested product is not shipped. These routine tests are performed on the raw materials as well as the manufactured medicines. By sourcing the microbe’s identity, the growth and metabolism can be investigated with the aim of finding an appropriate way to prevent it from growing any further and elimination; this is usually by recognizing the antibiotic which can destroy the cell membrane or the function of the particular pathogen. This can be done by isolating the strain of microbe and exposing it to various different antibiotics and observing and recording the different sensitivities; a popular method involving this is the disk diffusion method. Additionally, this process can reveal which strains of bacteria are resistant to certain sterilisation techniques and antibiotics. This is an important field of research in microbiology as antibiotic resistance is a common clinical issue that is faced today and is of growing concern in the pharmaceutical industry due to the fact that, every year, routinely used antibiotics are becoming redundant in the face of bacterial resistance (Toy, 2008). METHODS Refer to the four microbiology laboratory scripts: 1. Aseptic Technique and Examining the Environment 2. Identification 1 (Case Study) 3. Identification 2 4. Antibiotics RESULTS Table 1: Sterility Testing of Eyewash Samples Sample A B ✔ turbidity and deposits Growth C ✘ D ✔ deposits ✘ Table 2: Data from Differential and Selective Media Nutrient Agar Escherichia coli Sample A Sample C Proteus spp Sample A Sample C Salmonella Sample A Sample C Staphylococcus epidermidis Cream Colonies ✔ ✘ White and Spreading Description of Colony Forms CLED MacConkey’s Yellow, Opaque ✔ ✔ ✔ ✘ Translucent Blue ✘ ✘ Colourless ✘ ✘ White, Small Yellow, Small ✔ ✔ ✔ ✔ Sample A Sample C ✔ ✘ Colourless ✘ ✘ Cream Red, Mucoid Mannitol Salt No growth ✔ ✘ ✔ ✘ Flat Blue No growth Pink, Small ✘ ✔ 4/4 1/4 No growth ✘ ✘ ✘ ✘ 1/4 0/4 ✔ ✘ 2/4 0/4 Yellow to Clear, Very Small ✘ ✔ 2/4 4/4 Table 3: Data from the Gram Stain Eye Wash Sample Gram Stain (Positive or Negative) Organism Shape Does this support the tentative identification? A Negative (red) Rod Shaped ✔ C Positive (purple) Round/Circular ✔ Table 4: Presumptive Identification Eyewash Organism A Escherichia coli Probability Score /4 C Staphylococcus epidermis This is the presumed identity of the organisms due to the probability score shown in table 2 and the observations made in table 3. Table 5: API Results API Staph: 0 GLU FRU MNE MAL LAC TRE MAN XLT MEL NIT PAL VP - + + + + + - - - - + - - 6 7 0 RAF 6 XYL - - SAC MDG + 0 NAG - - ADH URE + + 1 LSTR 3 API 20 E: ONPG + ADH - LDC + ODC - CIT - 5 H2S - URE - 0 TDA - IND + VP - 4 GET - GLU + MAN - INO - 4 Organism Species A Escherichia coli Probability 99.2 % SOR + RHA SAC MEL AMY + + - 4 5 C Staphylococcus epidermidis 92.6% Definitive Identification: Eyewash Organism A Escherichia coli C Staphylococcus epidermis This definitive identification comes from a combination of the observed and probability results as well as the API results. Table 6: Identification of which Antibiotics are Effective at Killing the Two Bacterial Species Sample (Concentration) 10 5 2.5 1.25 X 0.5X 0.25X 0.125X Relative Concentration Log Known Sample Concentration (μg ml-1) 1 0 0.5 -0.3 0.25 -0.6 0.125 -0.9 Unknown Sample Concentration (μg ml-1) 1 0 0.5 -0.3 0.25 -0.6 0.125 -0.9 Average Zone Size (cm to 1 dp) 2.3 2.0 1.6 1.3 2.1 1.8 1.5 1.1 ARA OX + 2 1 Figure 1: A Log Dose-Response Curve Showing the Effect on the Log of Zone Inhibition against Log of Relative Concentration of a Known and Unknown Concentration of Gentamycin. M = 0.17 Antilog10 0.17 = 1.48 DISCUSSION The results obtained from the series of experiments all suggest that sample A is Escherichia coli and sample C is Staphylococcus epidermis. Escherichia coli is a bacterium which typically resides in the intestines and gastrointestinal tract of mammals, however it can be pathogenic and cause infection with symptoms including diarrhoea, stomach cramps and occasionally fever. In extreme cases, patients can develop haemolytic uremic syndrome (HUS), which can lead to kidney failure and death (NHS Choices, 2015). Staphylococcus epidermis is a bacterium which normally lives on the human skin and mucosa and has developed to become an “accidental” pathogen as it does not cause disease but rather acts host for resistant bacteria (Otto, 2010). Both of these bacteria are contaminants and therefore it is necessary to be able to identify them in the eye wash solutions as they can potentially cause harm to patients. In table 1, it is shown that only samples A and C showed growth of microbes whereas B and D remained a clear solution. This could be due to B and C having non selectivity to the liquid medium or, for the more probable reason, that they contained no bacteria. By using the four different mediums seen in table 2 – nutrient agar, CLED, MacConkey’s and mannitol salt – the identity of what type of microorganism was present in sample A and C could be solved via probability. As Escherichia coli is gram negative and Staphylococcus epidermis is gram positive, the gram staining results seen in table 3 is in line with the presumptive identification. The stain on sample A was red whereas C was purple. The purple colour is due to gram positive bacterial peptidoglycan cell wall retaining the crystal violet stain and the gram negative having a degraded outer membrane which is unable to hold this colour therefore is red (Toy, 2008). The bacteria structure also confirms the identities of sample A and C. The final aspect of the laboratories was to conduct an API test; by inoculating a plastic strip, which had 20 separate pockets, with a saline suspension of pure culture containing the specific dehydrated medium and observing the colour change, probability could be calculated by comparing it with a theoretical API reading table. Using the analytical profile, the probability of sample A being Escherichia coli and sample B being Staphylococcus epidermis was 99.2% and 92.6%, respectively. The lower percentage for sample B could be due to contamination of the culture. Figure 1 shows that the unknown concentration of gentamycin was less than the known concentration. The parallel lines show perfect correlation in linearity which shows that the assay is precise and the concentration of 1.48 M is accurate. The experiment could be improved upon by avoiding contamination by the use sterilisation techniques, such as autoclaving, wearing disposable gloves and using aseptic technique, which includes never leaving a culture dish open and minimising contact between equipment being used, e.g. sterile plastic loops and the micropipettes, and the bench surface. The agar diffusion method used in the final lab is a method used for monitioring antibiotic sensitivities and antibiotic levels in blood and tissues. It is a useful technique as it is easy and relatively cheap to carry out in a lab. However it has its limitations on a large scale; it is a slow and time consuming method as each diameter must be measured individually. This also brings in human error as diameters of that length and size have more of a percentage error when measuring. The method itself is not a true representation of body conditions therefore there is error associated in that respect too.