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Laboratory Portfolio
Report 1 - The Use of a Light Microscope to Identity Tissue Samples
Report 2 - The Use of Gram Staining Techniques to Determine Gram Negative andGram Positive Bacteria
Report 3 Antibiotic Susceptibility Testing Using the Disc Diffusion Method
STUDENT I.D 09151100[13/12/2009]
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CONTENTS
PAGE
The Use of a Light Microscope to Identify Tissue
Samples.......................................................... 4
ABSTRACT ...........................................................................................
...................................... 5
1.0
INTRODUCTION ....................................................................................
............................... 5
2.0
METHOD ..............................................................................................
................................ 5
3.0
RESULTS .............................................................................................
................................. 6
4.0
DISCUSSION ........................................................................................................................ 6
5.0
CONCLUSION .......................................................................................
............................... 6
REFERENCES
APPENDICES
The Use of Gram Staining Techniques to
Determine Gram Negative and Gram Positive
Bacteria ...................................................... 9
ABSTRACT ...........................................................................................
.................................... 10
1.0
INTRODUCTION.....................................................................................
............................. 10
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2.0
METHOD ..............................................................................................
.............................. 10
3.0
RESULTS............................................................................................................................. 11
4.0
DISCUSSION.........................................................................................
.............................. 11
5.0
CONCLUSION .......................................................................................
.............................. 12
REFERENCES
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CONTENTS cont.
PAGE
Antibiotic Susceptibility Testing
Using the Disc Diffusion
Method .........................................................................................
13
ABSTRACT ...........................................................................................
................................... 14
1.0
INTRODUCTION.....................................................................................
............................ 14
2.0
METHOD ..............................................................................................
............................. 14
3.0
RESULTS..............................................................................................
............................... 15
4.0
DISCUSSION.........................................................................................
.............................. 15
5.0
CONCLUSION .......................................................................................
.............................. 15
REFERENCES
APPENDICES
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FdSc Animal Science and Health Management
The Use of a Light
Microscope to IdentifyTissue Samples
Laboratory Portfolio : Report 1
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ABSTRACT:
This experiment shows the use of a light microscope as a method of tissue
sampling. The cellular structure of two different tissue samples was analysed andusing published documentation were able to carry out further research. From
these findings similarities were shown between the samples and the
documentation which led to the confirmation of the identity and location of the
tissue samples.
KEYWORDS: light microscope, tissue sampling, analyse, identify
1.0 INTRODUCTION:Tissue sampling refers to various procedures to obtain bodily fluids or tissue (e.g.
bone and muscle). The use of light microscopes in tissue sampling is an essential
part of analysing cellular structures and identifying whether there is any
infection, damage or disease present. The method describes here the use of a
light microscope to identify key features and structures in two tissue samples
(sample A, sample B), and the research techniques used to identify the tissue
type and its location.
2.0 METHOD:
First the light microscope was prepared by ensuring the power source was
turned on and that the source light was working. The next job to complete was to
clean the eyepiece and the objective lenses. This was done with a dry cloth but
could be done with a slightly moistened cloth if particularly dirty. It was
important to ensure that all parts were completely dry and not smeared before
use including the glass slide, so as not to affect any results. Before placing the
slide on the stage of the microscope the coarse adjustment knob was rotated
until the objective lens was about 2cms above the stage and the nosepiece was
rotated so that the low power objective lens was in line with the body piece.
Once the microscope was prepared, the slide was then clipped in place using the
stage clips. In order to view the image, the coarse adjustment knob was rotated
until the objective lens was about 5mm above the slide. Then using the fine
focus adjustment knob to fine focus the image, specific areas of the samples
were targeted. To look at the area in more detail the higher power lenses needed
to be used. The targeted area was moved into the centre of the stage, the nose
piece was rotated until the next objective lens clicked into place. The image
ought to have automatically focused but when this was not the case the fine
focus adjustment knob was used to refocus the image.
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3.0 RESULTS:
In sample A the first key feature that was identified was the folds or possible hair
like structure (fig.1). Specific areas of the sample were looked at by using the
higher power lenses and the cell structure was analysed. It was noted that the
sample had a stratified columnar appearance (fig.2). The same method was used
to analyse sample B and using the lower power lens there was a noticeable
folded structure (fig.3). By looking at specific areas using the high power lens it
was clear that the sample still had a stratified appearance but that the cell
structure itself was more cubiodal (fig.4). The differences between the samples
were initially minor and further investigation was needed to identify the location
of the two tissue type samples.
4.0 DISCUSSION:
To investigate further, published material was used to help identify the location
of the stratified columnar epithelium found in sample A and found that a hair like
appearance was also called ciliated. (P.R Wheater H. B., 1985). Further research
revealed that two main areas contained this fold like structure, the respiratory
system and the oviduct (fig.5 Medinfo, 2006). A photomicrograph of the oviduct
(fig.6 Medinfo, 2006) showed an image very similar to sample A, and after
looking at a range of images of the oviduct, found they were all were similar to
this sample. (P.R Wheater H. B., 1979, fig. 7 KU Medical Center, 2002). In
sample B, areas of tissue that contained stratified cubiodal epithelium were
invesigated. This intial investigation showed that the main function of this type of
cell was to act as a barrier but to also allow absorption, particulary in the duct
lining. (P.R Wheater H. B., 1985). Using electronic sources to gather additional
information of duct lining, it was publicised that a particular area showed an
image of an urethra exceptionally similar to sample B ( fig.8, Singh, T, 2006).
Further research showed that sample B also looked exceptionally similar to the
Ureter (P.R Wheater H. B., 1979).
5.0 CONCLUSION:
The identification of sample A was relatively straightforward. There was a range
of published material and images that confirmed sample A had come from the
Oviduct. For sample B further research was needed to try to identify whether the
sample came from the Urethra or the Ureter. The Urethra is the tube that carries
the urine from the bladder to the outside and the Ureters are the ducts that carry
urine from the kidneys to the bladder. As the cell structure of sample B allows
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absorption and was of a duct lining, it was concluded that the sample was that of
the Ureter.
REFERENCES:
K U Medical Center
www.kumc.edu/instruction/medicine/anatomy/histoweb/female (2002)
Medinfo http://medinfo.ufl.edu (2006)
P.R, Wheater, H.G, Burkitt, V.G, Daniels: Functional Histopathology (1979)
P.R, Wheater, H.G, Burkitt, A, Stevens, J.S, Lowe: Basic Histopathology (1985)
SinghT. ,Amarpal, Singh,R., Kinjavdekar P., Aithal H. P., Pawde A. M., Pratap K.
Indian Journal Of Veterinary Pathology: vol30 issue 20 Histopathological
Evaluation of Suture Materials and Tissue Adhesives for the Repair of Goat
Urethra (2006)
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http://www.kumc.edu/instruction/medicine/anatomy/histoweb/femalehttp://medinfo.ufl.edu/http://www.kumc.edu/instruction/medicine/anatomy/histoweb/femalehttp://medinfo.ufl.edu/ -
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APPENDICES:
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Fig.1 Sketch of sample A showing hairFig.2 Sketch of sample a showing stratified
Fig.3 Sketch of sample B showing noticeable
Fig.4 Sketch of sample B showing stratified
Fig.5 Ciliated columnar epithelium
Fig.6 Photomicrograph of Oviduct
Fig.7 Slide 10 Oviduct (K U MedicalFig.8 Image of Goat Urethra (Singh, T.
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FdSc Animal Science and Health Management
The Use of Gram
Staining Techniques toDetermine GramNegative and GramPositive Bacteria.Laboratory Portfolio: Report 2
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ABSTRACT:
The method showed the procedure used to identify gram positive and gram
negative bacteria. The results were further researched to confirm whether they
were gram positive and gram negative. It is vitally important to distinguish
between the two as they respond differently to antimicrobial agents and any
delay in the correct treatment could prove fatal.
KEYWORDS: gram positive, gram negative, identify, bacteria
1.0 INTRODUCTION:
Gram staining refers to a procedure used to determine whether particular
bacterial strains are positive or negative. Gram negative and gram positive
bacteria can be distinguished between by observing the ability of their cell wall
to stain with a dye called crystal violet. Gram positive bacteria will colour and
retain their colour when exposed to crystal violet, even when washed with
ethanol, whilst gram negative bacteria will lose their colour when washed with
ethanol, but will counter stain with safranin. This is of medical importancebecause gram positive and gram negative bacteria respond differently to
antibiotics. The method described here shows the procedure used to determine
gram negative and gram positive bacteria and the observations made whilst
analysing the samples.
2.0 METHOD:
To begin a Bunsen burner with a heat mat was needed. A wire inoculating loop
was sterilised by flaming it until it was red hot, then allowed it to cool. Using the
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sterilised inoculating loop, a drop of tap water was placed into the middle of a
clean glass slide. The loop was re sterilised and allowed it to cool. To take a
sample the neck of the culture bottle was flamed to sterilise. With the tip of the
loop, a small sample of the first bacterial culture (Staphylococcus Albus) was
removed, immediately the culture bottle was re flamed and closed to maintain
sterile conditions. The culture on the end of the inoculating loop was then mixed
with the water on the glass slide. Using a circular motion, the culture was spread
across the surface of the slide and the water, allowed to evaporate. Ensuring the
inoculating loop was dry, the loop was re flamed to sterilise. Putting a wet loop
into the Bunsen flame could have caused bacteria to have been released into the
air. To heat fix the smear, the glass slide was held with forceps and passed
quickly through the Bunsen flame. This was only done once as this could of
caused the smear to overheat and the bacteria to of been destroyed. Once this
was done, the Bunsen burner was turned off. Next taking a pipette of crystal
violet, over the sink, the smear was flooded. This was left for one minute before
pouring off the excess stain and gently washing the slide with distilled water. Thesmear was then flooded with Grams iodine solution and again left for one
minute. The excess stain was poured off and the remaining stain gently washed
off with a few drops of distilled water. This was followed by tilting the slide and
adding 6-8 drops of ethanol over a 2-3 second period, again this was washed it
off with distilled water. The next step was to flood the smear with safranin and
leave it for 15 seconds. As before, the excess stain was poured off and the
remaining stain gently washed with distilled water. The slide was then left to dry
naturally and the same method used to take a second culture sample (Spirillum
Serpens). Once completed, the smears were then observed under a light
microscope.
3.0 RESULTS:
Examining the Staphylococcus Albus was initially difficult. It appeared that too
much water had been applied to the slide and so much of the bacteria were not
visible. Using the higher power lenses specific areas of bacteria were looked at. It
was clearly stained purple indicating a gram positive bacteria. When analysing
the Spirillum Serpens bacteria it appeared that the same problem had occurred.Too much water had caused the bacteria to be sparse, making observations
difficult. Higher power lenses were used to target specific areas. Although
sparse, some bacteria were visible and appeared to be stained red. This
indicated that Spirillum Serpens were a gram negative bacteria. Further research
was carried out to confirm the results as there were some areas of purple
staining visible on the slide but no bacteria could be found.
4.0 DISCUSSION:
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Using the gram staining technique and observing under the light microscope is
vitally important in distinguishing Staphylococci from Streptococci.(Todar, K.
2008) Streptococci are gram negative so would require different antibiotics. The
infection would not respond to treatment unless the correct antibiotic was used.
Further investigation into Spirillum Serpens bacteria showed that they were helix
shaped and contained an outer membrane of two layers (Life, accessed 2009). It
is the absence of this outer membrane that allows the purple staining of gram
positive bacteria and the reason why gram negative bacteria can be counter
stained (Buckmire, F.L 1973). These investigations confirmed that Spirillum
Serpens bacteria are gram negative and the purple staining was a residue left on
the slide.
5.0 CONCLUSION:
It is important to ensure the procedure is followed correctly. On this occasion the
results were not clear as too much water had been added and so further
investigation was needed to confirm whether the smears were of gram positive
or gram negative bacteria. The examination of these results confirmed that
Staphylococcus was gram positive bacteria and Spirillum Serpens was gram
negative. This procedure is of vital medical importance in the treatment of many
infections and diseases. The wrong antibiotics could delay treatment, could make
the infection worse and even prove fatal.
REFERENCES:
Buckmire, F.L, Murray, R.G. Studies of the cell wall of Spirillum Serpens 11:
Characterisation of the outer structural layer. Journal of Bacteriology. 1976
Life Comparitive Characteristics of Gram Positive and Gram Negative Bacteria
accessed Nov 2009 www.life.umd.edu/classroom
Todar, Kenneth. Todars online Textbook of Bacteriology 2008
www.textbookofbacteriology.net/staph
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http://www.life.umd.edu/classroomhttp://www.textbookofbacteriology.net/staphhttp://www.life.umd.edu/classroomhttp://www.textbookofbacteriology.net/staph -
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FdSc Animal Science and Health Management
Antibiotic Susceptibility
Testing Using the DiscDiffusion Method.Laboratory Portfolio: Report 3
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ABSTRACT:
The method showed the procedure of antibiotic susceptibility using the disc
diffusion method to determine the optimal antimicrobial agent for three samples.
The results showed an error had occurred and the procedure would have to berepeated to obtain clear results. Determining the optimal antibiotic to treat an
infection or disease is of vital importance as inaccuracy could lead to fatality.
KEYWORDS: Susceptibility, identification, optimal, antibiotic
1.0 INTRODUCTION:
Antibiotics are compounds that damage bacteria. Antibiotic susceptibility testing
allows the identification of the optimal antibiotic to treat a particular bacterial
infection and is of great medical and economical importance. However it is also
important to treat diseases specifically to avoid the development of antibiotic
resistance. The method described here shows the use of aseptic techniques to
transfer three bacterial cultures and using the disc diffusion method, allows the
identification of the optimal antibiotic. The discs are impregnated with known
antimicrobial agents, this diffuses into the sampled medium causing a zone of
inhibited growth around the particular antimicrobial agent that it is susceptible
to.
2.0 METHOD:
To start this experiment a bottle of sterile nutrient agar was placed into a water
bath at 80 C to melt. At this point it was necessary to set up the Bunsen burner
with a heat mat. Once the agar had melted the bottle was removed and allowed
to cool to approximately 45 C. While the agar cooled a sterile inoculating loop
was needed. This was done by flaming it until it was red hot. The neck of the
non-pathogenic bacteria bottle was also flamed to sterilise it and using the
inoculating loop took a sample of the bacteria culture. This was transferred tothe bottle of cooled nutrient agar which was transferred to a Petri dish by lifting
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the lid to approximately 30 and immediately pouring the sample whilst swirling
the Petri dish. The Petri dish lid was then closed and the sample allowed to
solidify. Once solidified, sterile forceps were needed to transfer a mast ring to
the sample inside the Petri dish. The final step was to seal and label the Petri
dish and place it inside an incubator at 30 C. This method was repeated to take
another two samples. The samples were then left to incubate for seven days.
3.0 RESULTS:
Upon observation it was clear a problem had occurred. There was no evidence of
clearance zones around the mast ring on any of the three samples taken.
Bacteria growth had occurred, indicating that the samples had been takencorrectly and that the incubation time and temperature had not affected the
growth. This led to the conclusion that the mast ring may have been
compromised at some point or had been flawed.
4.0 DISCUSSION:
The mast ring indicates the susceptibility of the sample to the tested antibiotic
by a clear zone of inhibited growth around the impregnated paper discs (Huys,
2002). The diameter of the resulting zone (fig.1) allows the sample to becategorised as susceptible, intermediate or resistant to an antimicrobial agent
when comparing to international guideline tables. From the results obtained it
was not clear which antimicrobial agent any of the samples had been susceptible
to as there were no clear zones of inhibited growth around any of the discs.
5.0 CONCLUSION:
In this instance it would be necessary to repeat the procedure with a newconsignment of mast rings. It would be wise to take a number of samples to
compare results and would also help to confirm the correct antibiotic had been
found. Identifying the optimal antibiotic is of vital medical importance as
incorrect treatment could prove fatal. It is also vitally important that each strain
is tested individually as those continually exposed to the same antibiotic would
build up resistance. The disc diffusion method identifies the optimal antibiotic for
that specific strain of bacteria.
REFERENCES:
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www.healthhype.com/lab-tests-for-staph.htmlaccessed Dec 2009
Huys, Geert Antibiotic susceptibility testing of the aquaculture-associated
bacteria with the disc diffusion method. Laboratory of Microbiology, Unviversiteit
Gent. Nov 2002
APPENDICES:
Fig.1 Image showing the clearance zone around the optimal antibiotic on a
sample of Staphylococcus. (www.healthhype.com accessed Dec 2009)
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