Instructor’s name: Dr Andy, Dr David

Biology Laboratory Report

PRACTICAL #15, 16

Microbiology: Aseptic Techniques and Gram stain

Name: Nurgaliyeva Indira

Group G

Instructor’s name: Dr Andy, Dr David

Nazarbayev University

Date: April 8,15 2013

INTRODUCTION

Part A (Aseptic Techniques)

Microbiology is the study of different living microorganisms. In microbiology the study of different harmful bacteria or pathogens is impossible without especial safety precautions. Such precautiotechniques such aAseptic techniques are widely used in different areas. For example, in microbiology it is used to resist with different pathogens during inoculating the culture media (). Such techniques can be either traditional or modern methods. Moreover, they include the presence of the sterile gloves, masks instruments, disinfectors and antiseptics. The word antisepsis means the prevention of disease. Aseptic technique was firstly started by Joseph Lister in 1867.He was the first man who used antiseptics which are included to the aseptic techniques. In his experiment he used carbolic acid(phenol) to clean his workplace and one more equipment carbolic acid aerosol that is the good prevention to the harmful microorganisms. In AS sterilization stays in advance because it important to destroy the activity of all living microorganisms such as spores, bacteria, fungi and other. Autoclaves or the pressure cooker(Figure 1) is also used in the sterilization by cleaning the equipment before and after the experiments. Definitely the main thing people must do is to be careful to avoid the contamination of bacteria especially with harmful. There are two main types of the inhibition the bacteria: physical and chemical. Physical methods involve the heat, pressure, desiccation, radiation, sonic disruption and filtration. Chemical methods include range of different harmful and irritant chemicals. In microbiology aseptic techniques are popular in growing bacteria(Figure 2).

Figure 1. http://blog.universalmedicalinc.com/umi/autoclaves-the-questions-you-want-answered/

Figure 2. http://www.arabslab.com/vb/showthread.php?t=2728

In this practical the antibacterial properties of garlic (figure 3) and mint(figure 4) were investigated and compared. The antibacterial properties of garlic were discovered by Louis Pasteur in 1858, extracts of garlic inhibit the growth of several forms of pathogenic bacteria from the group of staphylococci, streptococci, bacillus and the cholera vibrio, as well as some pathogenic fungi. In the culture extracts of garlic toxic to tubercle bacilli. Antibacterial properties are mainly related to allicin. However, in subsequent years against these pathogens were found to be more effective antibiotics from other sources and their synthetic analogs. However, there is no reason to give up the garlic as an additional tool. Even Tear volatile garlic and irritation of nose and throat may benefit in inflammatory infections, including colds. These substances stimulate lacrimal gland and nasal cavity cancer, trachea and bronchi. They carry not only the secrets of washing these systems, facilitating breathing, but disinfection, due to the presence of secretions of glands special antibacterial enzyme lysozyme (lysozyme).

Mint - a very useful plant to humans. Mint essential oil contains (2.5%) whose principal ingredient is menthol, mint taste defines as well as other agents - esters felandren, pinene yasmon, piperitone, menthofuran, etc. There are a part of its tannins, flavonoids, bitter. Menthol mint makes a bactericidal plant. Mint has a lot of usufule properties, for example, analgesic and vasodilating. Decoction of the leaves of mint disinfect the mouth in various inflammatory processes. They relieve abdominal pain, heart pain, heartburn. Mint baths have soothing properties. Menthol - the main component of rmint essence. The latter is added in various toothpastes, powders, colognes, elixirs, etc.

Part B (Gram stain)

Gram staining procedure is a procedure used in microbiology to determine if the bacteria is gram positive or negative. It was firstly used by Danish physican H.C.J. Gram. The method of bacteria staining in which bacteria are stained dark purple (esspecialy Gram-positive bacteria), and in red or pink (gramotritsatelnye bacteria). The essence of the method is that the cell wall of Gram bacteria can hold the color even being bleached with ethanol, and therefore does not perceive additional color.In this case gram positive bacteria remain violet after being stained by crystal violet solution and then washed by ethanol where gram negative bacteria could not hold the color and were decolorized. This method is consider too be very usuful in classification of bacteria and in medicine. Range of gram-positive bacteria can be managed by penicillin or penn=icillin like antibiotics where gram-negative bacteria can response to a number of antibiotics.In this practicals aimed to microbiology antiseptic methods would be done and the determinetion of gram bacteria would managed.

Figure 3. http://www.kachestvo.ru/interesno/chesnok-poleznye-svoistva.html

Figure 4.http://mjata.ru/lechenie-mjatoj.html

AIM

Ø To practice the aseptic techniques;

Ø Be careful with reagents used in lab session

Ø To investigate the antibacterial properties of plants

SAFETY PRECAUTIONS

ü Wear safety goggles, lab coat and gloves;

ü Be careful with equipment, chemicals such as ethanol;

ü Investigation of antibacterial properties on bacteria must be dangerous that is why be carefully with opening the plates with bacteria;

ü At the beginning and at the end of the practical the hand must be washed and workplace should be cleared up and disinfected with ethanol;

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METHODS AND MATERIALS

Part A

Ø Agar plates must be prepared. This was done by TA.

Ø Plant extract was obtained and prepared with 10 cm³ of ethanol.

Ø Special antibiotic assay paper discs were used for each extract in this aseptic technique.

Ø With a help of sterile forceps 3 test discs with extract were put into bacterial plate together with suitable control. And underside the different discs were marked in order to be able to distinguish.

Ø Ethanol was used as a suitable control group.

Ø Petri dish then was closed and taped as shown on the picture.

Part B

Ø Cotton wool or wire loop was dipped into bacterial culture and then smeared a little onto a slide. The wool was disinfected by putting into the beaker.

Ø The slide was hold by forceps under the Bunsen flame until being disinfected

Ø As was shown on the picture in manual the slide must be placed under the sink

Ø The teat pipette was used and crystal violet solution and left for 1 minute. The excess of any stain was poured off and slide was gently washed by water.

Ø Then, iodine solution was poured onto the slide and left for 1 minute. A few drops of ethanol were used to wash the slide until no more violet stain appears.

Ø One more carbon Fuchsin was poured onto the slide and left for 15 seconds. Any excess of stain was poured off and water was used to wash the slide again.

Ø The microscope was used to view the stained bacteria. Blue/puprple= Gram-positive; red/pink= Gram-negative;

Ø Used slides were put into the disinfected beakers and the area was also disinfected by ethanol.

RESULTS

Part A

Antibacterial properties of plants on E.coli

Antibacterial properties of plants on B.sub

Part B

Discussion (Part A)

The veins after serving the organs take blood directly to the heart, whereas the veins which serve the alimentary canal take blood firstly to the liver, and then to the heart. This is because liver is the detoxification station, which purify and disinfects the blood from pollutes, toxicants and other harmful compounds. Rats are able to eat almost everything; therefore they have some features in their digestive system in order to perform its functions. Firstly, as all rodents, rats have well developed sharp and hard incisors. Thanks to these incisors rats are able to chew the hard substances, in order to receive the food. Secondly, it can be noticed on the drawings that there is no appendix in rats’ digestive system, and the size of the caecum is considerably large. This is due to the fact that they eat a lot of cellulose, and in order to digest it, the organism of rat has such a big caecum, which contains bacteria and participates in breakdown of the plant materials. Finally, rats do not have gall bladder. There some consequences of the fact of absence of appendix. For example, it is possible that rats’ organism suffers from several diseases, which entered the body with the food, because there is no appendix in their digestive system, which have an impact on immunity, because appendix has a lot of leucocytes. In general, the human and rat digestive systems are similar, because both of these species are vertebrates and mammalians. The location and presence of the same organs in both digestive systems make it similar. But there are several distinctions in the rat digestive system, because of the adaptation to the factors of living.

1. Respiratory system of the rat

Air enters the body through the nose, which is connected through the nasal and oral cavities to the pharynx. Air passes through the pharynx, whereas food passes to the esophagus. Air passes through the larynx to the trachea. Then the air goes to the bronchi, which divide to smaller sizes in the lungs, and finally to the alveoli, small sacs, which are totally permeated by capillaries. In the alveoli the process of absorption of oxygen by blood occurs, in the other words, oxygen enters the bloodstream.

Breathing is regulated by nervous and hormonal systems. Breathing center is located in medulla, and it contains two sections: inhalation and exhalation centers. Inner surface of breathing channels is covered with mucosa, which is made of ciliated epithelium. It has protective functions, because it excretes the protective mucus. The inhales and exhales are controlled by the diaphragm. During inhalation, the diaphragm contracts, thereby enhancing the thoracic cavity. Enlarging the cavity creates intake that pulls air into the lungs. While the relaxation of the diaphragm, air is exhaled by elastic recoil of the lung and the tissues lining the thoracic cavity in conjunction with the abdominal muscles.

Respiratory and alimentary tracts cross at throat, at the back side of the pharynx. Cross point, epiglottis controls passage of both air and food to trachea and esophagus, respectively. This allows several systems to function at the same time. For instance, human can eat and breathe, talk and breathe, vomit and breathe at the same time, because it would be impossible to survive without breathing. However, eating and talking together might lead to fatality. While talking epiglottis opens as the result, the bulk of food might jam in the respiratory tract, thus delaying the breathing process. Or sometimes when epiglottis is open air might go the stomach thus causing another problem such as inflation of the stomach.

2. Reproductive system of the rat

In this part of the laboratory work the excretory and reproductive systems of the rat were observed. The shape and general structure of these systems of the rat is similar to the human’s. This is also due to the fact that humans and rats are vertebrates and mammalians. Also we observed the differences in the structure of male and female reproductive systems. Differences between male and female reproductive systems are more clearly seen from both external and internal sides of the organism. Externally, male have scrotal sacs, inside of which testes are located, while female species have nipples. Furthermore, females have 3 separate for urinary, genetal openings and anus is the third, however in males, there is only 2, where 1 is for both urinary and genetal opening at the tip of the penis and second for digestive tract. Internally, the distinct feature of both species in that females have ovaries, while males have testes. The type of gamete they produce differ too, egg cells and sperm cells by female and male, respectively. Moreover, female get pregnant, and have organ called uterus, that carries baby. However, men do not give birth for offspring. Testes, located in scrotum, produce sperm cells, epidiymis stores them, Vas Deferens, prostate, seminal vesicles are glands that produce fluid. Furthermore, Cowper’s gland, penis and urethra are responsible for release of sperm.
The system in both genders is similar in developing from one embryonic tissue. Both species have gonads producing gametes.

Q4. Respiratory and alimentary tracts cross at throat, at the back side of the pharynx. Cross point, epiglottis controls passage of both air and food to trachea and esophagus, respectively. This allows several systems to function at the same time. For instance, human can eat and breathe, talk and breathe, vomit and breathe at the same time, because it would be impossible to survive without breathing. However, eating and talking together might lead to fatality. While talking epiglottis opens as the result, the bulk of food might jam in the respiratory tract, thus delaying the breathing process. Or sometimes when epiglottis is open air might go the stomach thus causing another problem such as inflation of the stomach.
Q5. To begin with, differences between male and female reproductive systems are more clearly seen from both external and internal sides of the organism. Externally, male have scrotal sacs, inside of which testes are located, while female species have nipples. Furthermore, females have 3 separate for urinary, genetal openings and anus is the third, however in males, there is only 2, where 1 is for both urinary and genetal opening at the tip of the penis and second for digestive tract. Internally, the distinct feature of both species in that females have ovaries, while males have testes. The type of gamete they produce differ too, egg cells and sperm cells by female and male, respectively. Moreover, female get pregnant, and have organ called uterus, that carries baby. However, men do not give birth for offspring. Testes, located in scrotum, produce sperm cells, epidiymis stores them, Vas Deferens, prostate, seminal vesicles are glands that produce fluid. Furthermore, Cowper’s gland, penis and urethra are responsible for release of sperm.
The system in both genders is similar in developing from one embryonic tissue. Both species have gonads producing gametes.

Q6. The reason why female species have three holes in their ability to give birth to their offsprings, baby comes out from the middle hole, while the rest of holes are responsible for ordinary functions as in males. However males have only two holes, urinary and genetal openings are in one hole. Consequently, urethra is elongated according to penis. Elongation evolved to fertilize the egg cells, thus enabling to continue the gender.

Validity of results

The results are valid because all the experiments are repeatable. At the same time results can not be totally valid due to different reasons. Firstly, because in experiment uncertain and different amount of bacteria and plant extract were used. That is why student got different results. Moreover due to the some errors bacteria could be incompletely spread into the agar plate that is why the antibacterial discs with the extract could be placed into the place without bacteria.

Errors

There are more random than systematic errors. Due to the shortage knowledge of bacteria and specific rules aseptic technique can be wrongly hold. Bacteria could be incompletely spread into the agar plate that is why the antibacterial discs with the extract could be placed into the place without bacteria. The plant extract could be incompletely pounded and the main antibacterial compounds could be missed out.

Limitation

Limited amount of staining solutions were given that is why it was essential to be accurate during the second part of lab session. Moreover only two types of bacteria were given in order to avoid the contamination of harmful pathogens and its spreading into the our organisms.

CONCLUSION

In this lab session two labs based on the new topic called microbiology were managed in order to investigate the bacterial properties of plant extract, to compare their properties and mainly to be familiar with aseptic techniques. Aseptic techniques are very useful in working with bacteria. It was deduced from the experiment that garlic and mint extract can really kill the bacteria. Due to the large diameter of the disinfected area Ethanol that was used as a control group is the best bacteria killer in contrast with garlic and mint.

REFERENCE

1.Roberts, Michael J. Reiss, Grace Monger.2000. Advanced Biology. China: Midas Printing International Ltd.

2.Engelkirk, P. G., Duben-Engelkirk, J., & Burton, G. R. W. 2011. Burton's microbiology for the health sciences. Lippincott Williams & Wilkins.

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