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Preet Sidhu 1299064 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation Title: Case Study of Pathogens

24 May 2013

Aim: To investigate the wound for the colonial and gram stain characteristics of pathogenic skin flora. Materials & Methods: As given on page no. 28 & 29 on MICROBIOLOGY B- Practical Manual. Results: Sample details: Name: Marcus Doolittle , D.O.B: 1.1.1995, Sample Taken at 8:3p am on 18th April 2013-05-18 ,Sample type: Knee wound Fig 1: Wound Swab

HBA- Aerobically White & cream colour colonies Small and biz size colonies Haemolysis Smooth & Shiny texture 2 different types of colonies (cream & white cream colour colonies)

Fig 2: Wound organism on HBA (Aerobically)

Small colonies (cream colour) - haemolysis Big colonies (cream white Colour)

Fig 2: The organism was inoculated from wound swab on Horse Blood Agar (HBA) HBA- Anaerobically

Preet Sidhu 1299064 24 May 2013 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation 2 different types of colonies. More small colonies /less large colonies. Cream colour colonies Smooth and shiny texture - Haemolysis Figure3: Wound Organism on HBA (Anaerbically) - Haemolysis

Small colonies (cream colour)

Big colonies (cream white Colour)

Fig 3: The organism was inoculated from wound swab on Horse Blood Agar (HBA) Growth on MAC Smooth and shiny texture Small and big colonies, two types of colonies Light purple discrete colonies ( gram positive coccus) Dark purple colonies (Gram negative bacillus) Rough texture

Fig 4: Inoculation of wound sample on MacConkey Agar

Lactose fermenter colonies

Preet Sidhu 1299064 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation

24 May 2013

From the above plates, purity plates were prepared. Colonies were inoculated from HBA aerobically and anaerobically plates on TSA by splitting the plate into two parts. PURITY PLATES: Bigger colonies: Smooth and shiny texture Round in shape Mixed colonies observed Small colonies predominant than large size colonies White cream indicates small colonies Cream yellow indicates large colonies. Smaller colonies: Smooth and shiny texture Dense and mixed colonies on plate Small discrete colonies (white cream colour) Large colonies (cream yellow colour)

Fig 5: Purity Plate from HBA (Aerobically)

Big Size colonies

Small size colonies

Fig 5: Both types of colonies (small & big colonies) inoculated on TSA. The resulted colonies were mixed colonies as seem on plates. HBA (Aerobically) - Big size colonies on TSA Mixed colonies Discrete small white cream colour colonies Small number of large colonies

Preet Sidhu 1299064 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation HBA (Aerobically) Small size colonies on TSA Dense and mixed colonies. Small white colour colonies Smooth and shiny texture Fig 6: HBA (Aerobically) on TSA

24 May 2013

Small Size colonies -inoculation area

Big Size colonies -inoculation area

Fig 6: Colonies from HBA (O2) on TSA. It was observed that the colonies were mixed. HBA (Anaerobically) Big colonies on TSA Smooth and shiny texture Dense and mixed colonies Discrete small white cream colour colonies Small number of large colonies Fig 7: Colonies from HBA (Anaerobically) on TSA Fig 7: Both types of colonies Big Size colonies from -inoculation HBA area

Small Size colonies -inoculation area (Anaerobically) inoculated on TSA and the resulted colonies were mixed colonies.

Preet Sidhu 1299064 24 May 2013 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation GRAM STAIN RESULTS: The mixed colonies were selected from both aerobically and anaerobically HBA plates. Gram positive cocci (Bigger colonies) Purple in colour Background clear Cocci in shape

Clear background

Purple in colour and round in shape

Diagram 1: Gram stain appearance under microscope Smaller colonies Purple in colour Background clear Cocci in shape Diagram 2: Gram stain appearance under microscope

of big size colonies

of big size colonies

Preet Sidhu 1299064 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation

24 May 2013

Clear background

Purple in colour and round in shape

Fig 8: Gram stain Results of Bigger Colonies

Purple in colour and round in shape Background clear

Identification of organism: Gram stain test helps to which test perform next. According to the dichotomous key, catalase test is the first option for the identification.

Preet Sidhu 1299064 24 May 2013 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation Both small and large colonies were used for catalase test. The 2-3 big size colonies were picked from plate and put on the slide. A drop of H2O2 was put on the cover slip. Then cover slip was placed on the slide. The H2O2 reacts with organism and starts producing bubbles. TABLE 1: Catalase test result Colony type Big size colony Small size colony Result Bubbles produced Positive result Bubbles produced Positive result

COAGULASE TEST: Organism starts clotting when it mixes with rabbit plasma. It shows positive results. Coagulase test indicates the infection is caused by Staphylococcus aureus. Discussion: The clinical gram stain results provide a information that wound contains 3+ pus cells, 3+ Gram positive cocci, 1+ Gram negative bacilli. 3+ pus cells indicate that wound have definitely infection which is mostly caused by 3+ gram positive cocci species. The 3+ number of Gram positive cocci also represent that it is causative organism. Most of the wound infection is caused by S.aureus and S.pyogens (GPC) and E.coli, K.pneumoniae. K.pneumoniae and E.coli is present as a result of environmental contamination. The wound sample was collected by using transport swab. Transwab is the transport swab with medium that is suitable for both aerobes and anaerobes organism. The transwab is inert to microorganisms which aids in the recovery of delicate organisms. It does not allows over grow and high pathogens. The next step was isolating the organism by growing on HBA and MAC media. The organism was inoculated on HBA both aerobically and anaerobically conditions. In aerobic conditions, it shows haemolysis (incomplete) and 2 different types of colonies were observed on plates which were differentiating by the size of colonies (fig 2) . On the other hand, -haemolysis was observed in anaerobically conditions. The colony morphology was same as aerobic colony morphology. 2 different types of colonies were observed on the plates. The morphology of colonies can be described on the basis of size. (Fig 3) The bigger colonies were about 2-3mm and smaller colonies were about 0.5- 1mm. The wound sample was also inoculated on the MAC media. As MAC agar inhibits the growth of gram positive bacteria and only allow to gram negative bacillus to grow on media. The colonies were having rough texture which shows that the organism doesnt ferment lactose. Only 4-5 colonies shows lactose fermentation (Fig 4)

Preet Sidhu 1299064 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation

24 May 2013

Identification: After isolation of organism on the basis of size, it allows to perform the identification step. For the identification, the purity plates are very important as purity plates provide a confidence about purity of the culture that is used for further identification process and it also confirms that there is no contamination occurs in sample. Purity plates were prepared and the results on colony morphology are given in the results section. The organism from HBA, both aerobically and anaerobically was inoculated on TSA plates by splitting in half way and incubated at 350C for 24 hours. The plates were examined after 24 hours. Mixed dense colonies were observed on the plates. The colonies were clearly differentiated on TSA on the basis of size (small colonies about 0.5-1mm and big colonies- about 2-3mm). The reason for getting mixed colonies was that too much colonies were inoculated on the TSA-half spilt plate. As it is half split, it needs small amount of colonies to inoculate for getting single colonies. The predominant colonies were of small size as it was much numerous in amount as compared to larger size colonies. Both small and biz size colonies used for gram stain. The gram stain results show that both organisms were gram positive cocci (Fig 8) species as under microscope it was purple in colour and round in shape. The gram stain results allow further testing on gram positive cocci. According to dichotomous key of GPCs test, the first test is catalase test. Both organisms were catalase positive which ultimately leads to next step which was coagulase test. Coagulase test was only performed on the mixed colonies that were inoculated on TSA purity plate that was made for Rapid Staph. Test. This test was not performed as it doesnt work according to the expectation of supervisor. Rabbit plasma was used for the coagulation test. The colonies were mixed with rabbit plasma, it starts to coagulase (clotting) (white bubbles seen). As the rabbit plasma was very old, so it doesnt clot properly. The supervisor examines the colony morphology on plates and confirms that it was coagulase positive organism. From the dichotomous key of identification, it was found that Staphylococcus aureus is the causative organism for the wound infection. It also matches with clinical gram stain results which also show 3+ GPC were causing infection. According to the clinical gram stain, it also shows that 1+GNB were present, it may be present in the wound as some of colonies observe on the MAC agar media but didnt observe in results because of the mixed colonies. The infection is local at this stage but if it is not treated with proper antibiotics the infection will become systemic. ABOUT CAUSTIVE ORGANISMS: S.aureus is gram positive cocci, facultative anaerobic bacteria. It is catalase positive which indicates that it able to produce enzyme catalase, so it is able to convert hydrogen peroxide to H2O and O2. It also produces coagulase enzyme which helps to clot the plasma and prevent the bacterial

Preet Sidhu 1299064 24 May 2013 Unit: Microbiology B Course: Bachelor Of Biotechnology and Innovation cell from phagocytosis. The virulence factors of S.aureus include enzymes, antigens, capsules and toxins help to cause a infection or disease. Errors: Large amount of colonies inoculated on half spilt TSA which gives results of mixed and dense colonies on plate. So it is important to take less amount of single colonies to get better results. Conclusion: The purpose of the testing to examine the wound and identify which organism is causative organism for wound infection. Gram stain provides the information that the organism was gram positive cocci. After performing catalase and coagulase test, Staphylococcus aureus was found as the causative organism for the wound infection. Acknowledgements: I would like to say my special thanks of gratitude to my teacher Robyn Megna who held a practical sessions to enhance the understanding of pathogens and help the students during the experiment session. Reference: Rapid Microbiology, NA, Microbiological Swabs - Capture, Maintain and Release!, retrieved on 18/05/2013 from http://www.rapidmicrobiology.com/test-methods/Swabs.php Thermofisher, NA, Transwab, retrieved on 18/05/2013 from http://www.thermofisher.com.au/Uploads/file/Scientific/MicrobiologyProducts/Microbial-Safety-Testing/Swabs/MWE/MWE-Transwab.pdf Wikipedia, the free encyclopaedia, 2013, Staphlococcus aureus, retrieved on 18/05/2013 from http://en.wikipedia.org/wiki/Staphylococcus_aureus