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Microbiology lab report
Laboratory Skills
University of Portsmouth
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An experiment using the aseptic technique to cultivate Staphylococcus aureus ( S. aureus) on an agar plate to observe the isolation of its respective colonies and to record observations of 10 microorganisms under a microscope.
Introduction
Cultivation of bacteria is important in healthcare, it is the process of multiplying microorganisms in a culture media under controlled laboratory conditions 1. In hospitals, it is used to separate bacteria from DNA samples of patients to determine what infection they may have. This is performed by a process called streaking. Streaking isolates colonies of bacteria so that they can be visualised under a microscope and properly identified 2. This must be done in aseptic conditions 3 in order to prevent contamination from pathogens.
In our experiment, we aimed to practise streaking and microscopic visualisation under aseptic conditions, produce a streak plate with isolated colonies and to determine the correct Gram stain colour, shape and arrangement of 10 microorganisms.
Method
First, we prepared an agar plate. We took universal containers (20cm 3 ) of nutrient agar (melted at 98° and cooled to 56°) from the water bath, transferred it to a sterile Petri dish (labelled at the base) and left to set for 10 minutes.
We sterilised an inoculating loop in a Bunsen burner until it was red hot along the whole wire and waited to cool slightly. The loop was then inserted halfway into the bottle of culture and the cap was securely replaced back on the bottle. The organisms were spread across the agar plate and then returned to its lid. The loop was re-sterilised in the Bunsen burner slowly and cooled, then produced further streaks from the point of inoculation (Figure 1). The plate was put in an incubator (37°) for 48 hours. We observed the colonies and drew a model. Microscope slides were observed of different bacteria prepared by bacterial smearing and staining. We recorded their Gram stain colour, their bacterial shape and arrangement.
Results
Our results from Table 1 show the Gram stain colour of each microorganism after being stained by safranin to determine if they are Gram positive or negative. The shape and arrangement was also observed to identify the microorganism further.
Discussion
Streaking is the process of isolating single colonies of an organism on an agar plate 2 , therefore what we expected to observe was isolated, single colonies of our culture in the fourth quadrant. The microorganisms seemed to become more isolated after each quadrant on the agar plate, which can be observed in Figure 2, therefore is what was expected. This overall shows a positive outcome on the streaking technique. Most of our results were correct, since they complied with a known table 5. The exception was Escherichia coli as they have a cocci shape 8 , instead of our recorded bacilli shape (Table 1). This was unclear to see under the microscope due to the fact that this microorganism’s cells were very closely packed together therefore difficult to identify. To overcome this, we could’ve observed the edge of the slide, where the cells were less closely packed together, and used a higher resolution to identify the shape further.
Gram staining separates bacteria into two groups dependant on their cell wall composition 5. The lipopolysaccharide membrane in Gram negative cells is degraded after the addition of ethanol 7 and the thin peptidoglycan cell wall can’t retain the crystal violet-iodine complex, formed from the crystal violet die and iodine, so the colour is lost 5 ; it is the counterstain safranin that gives Gram negative cells their pink colour. The peptidoglycan cell wall in Gram positive bacteria is dehydrated due to numerous teichoic acid cross-links, therefore it tightens and traps the crystal violet-iodine complex, so remains a purple colour.
Gram staining results can vary due to cell wall damage, over-decolourisation of the smear or if the procedure was performed from an old culture 6. If the smear is too thick, it can also be difficult to recognise the decolourisation of the Gram negative bacteria 6. I didn’t experience any of these variations since my Gram stain results complied with a known table 5.
References
Cliffsnotes. (2017). Microbial Cultivation. [online] Available at: cliffsnotes/study-guides/biology/microbiology/microbial- cultivation-and-growth/microbial-cultivation [Accessed 18 November 2017].
Nuffieldfoundation. (2017). Making a streak plate [Nuffield Foundation] Available at: nuffieldfoundation/practical-biology/making-streak- plate [accessed 18 November 2017].
Kristeen Cherney and Rachell Nall (Heathline) 2017 Aseptic Technique. Available from: healthline/health/aseptic-technique#overview1 [18 November 2017]
Pinsdaddy. (2017). Staphylococcus Aureus On Agar Pictures to Pin on Pinterest page 12 - PinsDaddy. [online] Available at: pinsdaddy/staphylococcus-aureus-on-
Microbiology lab report
Module: Laboratory Skills
University: University of Portsmouth
