wound case study

7/30/2019 Wound Case Study

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Preet Sidhu 1299064 24 May 2013

Unit: Microbiology B

Course: Bachelor Of Biotechnology and Innovation

Title: Case Study of Pathogens

Aim: To investigate the wound for the colonial and gram staincharacteristics of pathogenic skin flora.

Materials & Methods: As given on page no. 28 & 29 onMICROBIOLOGY B- Practical Manual.

Results: Sample details:Name: Marcus Doolittle , D.O.B: 1.1.1995, Sample Taken at 8:3p amon 18th April 2013-05-18 ,Sample type: Knee woundFig 1: Wound Swab

HBA- Aerobically

White & cream colour colonies Small and biz size colonies

α – Haemolysis

Smooth & Shiny texture

2 different types of colonies (cream & white cream colourcolonies)

Fig 2: Wound organism on HBA (Aerobically)

Fig 2: The organism was inoculated from wound swab on Horse

Blood Agar (HBA)HBA- Anaerobically

Big colonies (cream white

Colour)

Small colonies (creamcolour)

α- haemolysis






2 different types of colonies.

More small colonies /less large colonies.

Cream colour colonies

Smooth and shiny texture

β- HaemolysisFigure3: Wound Organism on HBA (Anaerbically)

Fig 3: The organism was inoculated from wound swab on HorseBlood Agar (HBA)

Growth on MAC


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

β- Haemolysis

Big colonies (cream white

Colour)

Small colonies (cream

colour)

Lactose fermenter

colonies






From the above plates, purity plates were prepared. Colonies wereinoculated from HBA aerobically and anaerobically plates on TSA bysplitting the plate into two parts.

PURITY PLATES:

Bigger colonies:


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:


Dense and mixed colonies on plate

Small discrete colonies (white cream colour)

Large colonies (cream yellow colour)

Fig 5: Purity Plate from HBA (Aerobically)

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

Small size colonies

Big Size colonies






HBA (Aerobically) – Small size colonies on TSA

Dense and mixed colonies.

Small white colour colonies

Smooth and shiny textureFig 6: HBA (Aerobically) on TSA

Fig 6: Colonies from HBA (O2) on TSA. It was observed that thecolonies were mixed.HBA (Anaerobically) – Big colonies on TSA


Dense and mixed colonies

Discrete small white cream colour colonies

Small number of large coloniesFig 7: Colonies from HBA (Anaerobically) on TSA

Fig 7: Bothtypes of colonies

from HBA

(Anaerobically) inoculated on TSA and the resulted colonies weremixed colonies.

Small Size colonies

-inoculation area

Big Size colonies

-inoculation area

Small Size colonies

-inoculation area

Big Size colonies

-inoculation area






GRAM STAIN RESULTS: The mixed colonies were selectedfrom both aerobically and anaerobically HBA plates.

Gram positive cocci (Bigger colonies)

Purple in colour

Background clear

Cocci in shape

Diagram 1: Gram stain appearance of big size coloniesunder microscope

Smaller colonies

Purple in colour

Background clear

Cocci in shape

Diagram 2: Gram stain appearance of big size coloniesunder microscope

Purple in

colour and

round in shape

Clear

background






Fig 8: Gram stain Results of Bigger Colonies

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

Purple in

colour and

round in shape

Clear

background

Purple in

colour and

round in shape

Background

clear






Both small and large colonies were used for catalase test. The 2-3big size colonies were picked from plate and put on the slide. A dropof H2O2 was put on the cover slip. Then cover slip was placed on theslide. The H2O2 reacts with organism and starts producing bubbles.

TABLE 1: Catalase test result

Colony type ResultBig size colony Bubbles produced – Positive

resultSmall size colony Bubbles produced – Positive

result COAGULASE TEST: Organism starts clotting when it mixes withrabbit plasma. It shows positive results.

Coagulase test indicates the infection is caused by Staphylococcusaureus.

Discussion: The clinical gram stain results provide a informationthat wound contains 3+ pus cells, 3+ Gram positive cocci, 1+ Gramnegative bacilli. 3+ pus cells indicate that wound have definitelyinfection which is mostly caused by 3+ gram positive cocci species. The 3+ number of Gram positive cocci also represent that it iscausative organism. Most of the wound infection is caused byS.aureus and S.pyogens (GPC) and E.coli, K.pneumoniae.

K.pneumoniae and E.coli is present as a result of environmentalcontamination. The wound sample was collected by using transportswab. Transwab is the transport swab with medium that is suitablefor both aerobes and anaerobes organism. The transwab is inert tomicroorganisms which aids in the recovery of delicate organisms. Itdoes not allows over grow and high pathogens.

The next step was isolating the organism by growing on HBA andMAC media. The organism was inoculated on HBA both aerobicallyand anaerobically conditions. In aerobic conditions, it shows α-

haemolysis (incomplete) and 2 different types of colonies wereobserved on plates which were differentiating by the size of colonies(fig 2) . On the other hand, β-haemolysis was observed inanaerobically conditions. The colony morphology was same asaerobic colony morphology. 2 different types of colonies wereobserved on the plates. The morphology of colonies can bedescribed on the basis of size. (Fig 3) The bigger colonies wereabout 2-3mm and smaller colonies were about 0.5- 1mm. Thewound sample was also inoculated on the MAC media. As MAC agarinhibits the growth of gram positive bacteria and only allow to gramnegative bacillus to grow on media. The colonies were having rough

texture which shows that the organism doesn’t ferment lactose.Only 4-5 colonies shows lactose fermentation (Fig 4)






Identification: After isolation of organism on the basis of size, itallows to perform the identification step. For the identification, thepurity plates are very important as purity plates provide aconfidence about purity of the culture that is used for furtheridentification process and it also confirms that there is nocontamination occurs in sample. Purity plates were prepared andthe results on colony morphology are given in the results section. The organism from HBA, both aerobically and anaerobically wasinoculated on TSA plates by splitting in half way and incubated at350C for 24 hours. The plates were examined after 24 hours. Mixeddense colonies were observed on the plates. The colonies wereclearly differentiated on TSA on the basis of size (small colonies –about 0.5-1mm and big colonies- about 2-3mm). The reason forgetting mixed colonies was that too much colonies were inoculated

on the TSA-half spilt plate. As it is half split, it needs small amountof colonies to inoculate for getting single colonies. The predominantcolonies were of small size as it was much numerous in amount ascompared to larger size colonies. Both small and biz size coloniesused for gram stain. The gram stain results show that bothorganisms were gram positive cocci (Fig 8) species as undermicroscope it was purple in colour and round in shape. The gramstain results allow further testing on gram positive cocci. Accordingto dichotomous key of GPC’s test, the first test is catalase test. Bothorganisms were catalase positive which ultimately leads to nextstep which was coagulase test. Coagulase test was only performed

on the mixed colonies that were inoculated on TSA purity plate thatwas made for Rapid Staph. Test. This test was not performed as itdoesn’t work according to the expectation of supervisor. Rabbitplasma was used for the coagulation test. The colonies were mixedwith rabbit plasma, it starts to coagulase (clotting) (white bubblesseen). As the rabbit plasma was very old, so it doesn’t clot properly. The supervisor examines the colony morphology on plates andconfirms that it was coagulase positive organism. From thedichotomous key of identification, it was found that Staphylococcusaureus is the causative organism for the wound infection. It also

matches with clinical gram stain results which also show 3+ GPCwere causing infection. According to the clinical gram stain, it alsoshows that 1+GNB were present, it may be present in the wound assome of colonies observe on the MAC agar media but didn’t observein results because of the mixed colonies. The infection is local at thisstage but if it is not treated with proper antibiotics the infection willbecome systemic.

ABOUT CAUSTIVE ORGANISMS: S.aureus is gram positive cocci,facultative anaerobic bacteria. It is catalase positive which indicatesthat it able to produce enzyme catalase, so it is able to convert

hydrogen peroxide to H2O and O2. It also produces coagulaseenzyme which helps to clot the plasma and prevent the bacterial






cell from phagocytosis. The virulence factors of S.aureus includeenzymes, antigens, capsules and toxins help to cause a infection ordisease.

Errors: Large amount of colonies inoculated on half spilt TSA whichgives results of mixed and dense colonies on plate. So it isimportant to take less amount of single colonies to get betterresults.

Conclusion: The purpose of the testing to examine the wound andidentify which organism is causative organism for wound infection.Gram stain provides the information that the organism was grampositive cocci. After performing catalase and coagulase test,Staphylococcus aureus was found as the causative organism for thewound infection.

Acknowledgements: I would like to say my special thanks of gratitude to my teacher Robyn Megna who held a practical sessionsto enhance the understanding of pathogens and help the studentsduring the experiment session.

Reference:Rapid Microbiology, NA, Microbiological Swabs - Capture, Maintainand Release!, retrieved on 18/05/2013 fromhttp://www.rapidmicrobiology.com/test-methods/Swabs.php

Thermofisher, NA, Transwab, retrieved on 18/05/2013 fromhttp://www.thermofisher.com.au/Uploads/file/Scientific/Microbiology-Products/Microbial-Safety-Testing/Swabs/MWE/MWE-Transwab.pdf

Wikipedia, the free encyclopaedia, 2013, Staphlococcus aureus,retrieved on 18/05/2013 fromhttp://en.wikipedia.org/wiki/Staphylococcus_aureus

http://www.rapidmicrobiology.com/test-methods/Swabs.php

http://www.thermofisher.com.au/Uploads/file/Scientific/Microbiology-Products/Microbial-Safety-Testing/Swabs/MWE/MWE-Transwab.pdf


http://en.wikipedia.org/wiki/Staphylococcus_aureus

http://www.rapidmicrobiology.com/test-methods/Swabs.php



http://en.wikipedia.org/wiki/Staphylococcus_aureus

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