Laboratory Portfolio Student I.D 09151100

8/14/2019 Laboratory Portfolio Student I.D 09151100

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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]

2204 WORDS


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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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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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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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http://www.healthhype.com/lab-tests-for-staph.htmlhttp://www.healthhype.com/http://www.healthhype.com/lab-tests-for-staph.htmlhttp://www.healthhype.com/

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