Etiket Arşivleri: Nutrient broth

Lab Procedures Part I. Staining and Colony Morphology

Ex. 1-1 Types of Media

Nutrient agar and nutrient broth are common media used in maintaining bacterial cultures. They contain nutrients such as proteins, carbohydrates (sugars), and various lipids. Solid media contains agar, a thickening agent that does not melt at high temperatures (this allows us to incubate the bacteria to facilitate growth); bacteria grow on the surface of solid media. Preparation of media involves making solutions and then autoclaving them at 121oC, 15 psi, for 15 minutes to kill all bacteria including thermophiles (heat loving bacteria) and those that produce resistant endospores (the increased pressure gets the temperature above boiling). This will insure that the media is sterile before inoculation. After autoclaving, media can be dispensed into petri dishes/test tubes and cooled.

Examine different kinds of media: nutrient broth tubes, nutrient agar slants, nutrient agar deeps, agar plates

What would be the advantage of growing bacteria on a slant, rather than on a deep?

Ex. 2-1 Ubiquity

Work in groups of 4

Each group needs 4 agar plates

Label each plate on edge of plate with: name, date, experiment #, type of exposure

Plate 1 – open and place on top of bench; expose to air for 30 minutes.

Plate 2 – Divide plate in half (write on bottom of plate with sharpie marker; label one half of plate with a “B” (before);

label the other half with an “A” (after); touch agar on “B” side with a dirty finger; wash hands with antibacterial soap, then touch agar on “A” side with clean finger

Plate 3 – kiss agar

Plate 4 – dip a sterile swab in sterile water (found in test tubes); swab bench top; dispose of swab in trash.

Incubate: Plates 1 & 4 at 30oC; Incubate: Plates 2 & 3 at 37oC.

Ex. 3-1 Introduction to the Light Microscope

Read/discuss Intro. Microscope Handout

Clean microscope before viewing slides


Culture Formulations

Adi Sıvı Besi Yeri(Adi Buyyon)

Bileşimi: Yağsız sığır kıyması………………………500 g
Pepton………………………………………10 g pH= 7.2-7.4
Tuz (NaCl)……………………………………5 g
Su……………………………………………1000cc(L)
Hazırlanışı:500g yağsız sığır kıymasına 1 L saf su konur ve serin bir yerde 2 saat bekletilir. Bir saat yavaş yavaş karıştırılarak kaynatılır. Süzülür. Sıcakken pepton ve tuz eritilir. Eksilen su kadar saf su konup litreye tamamlanır. pH’sı 7,2–7,4’e ayarlanır. Deney tüplerine 15 ml kadar dağıtılır. Tüplerin ağızları pamuklanıp, 121 ºC’ta 15 dakika sterilize edilir

Katı Besi Yeri(Adi Agar)

Bileşimi: Adi Buyyon…………………………………1000cc(L)
Agar………………………………………… 15 g pH =7.2-7.4
Hazırlanışı: Cam balondaki adi buyyona tartılan agar katılır. Agar eriyene kadar karıştırılarak kaynar su banyosunda tutulur. pH’ı 7.2–7.4’e ayarlanır. Balonun ağzı pamuklanıp, otoklavda 121 ºC’ta 15 dakika sterilize edilir. Sıcakken, besi yeri henüz katılaşmadan steril petri kutularına 15–20 ml boşaltılıp katılaşması beklenir.

Nutrient Broth: 

Bakterilerin gelişimi için kullanılan genel sıvı besi yeridir.
Bileşimi:1) Et ekstraktı……………………………………1 g
Maya ekstraktı………………………………..2 gr pH= 7.4 ±0.2
Pepton…………………………………………5 g
Tuz (NaCl)……………………………………5 g
Saf su………………………………………..1000cc(L)
Bileşimi:2) Et ekstraktı……………………………………10 g
Pepton…………………………………………10 gr pH 7.5±0.2
Tuz (NaCl)……………………………………5 g
Saf su………………………………………..1000cc(L)
Bileşimi:3) Et ekstraktı……………………………………3 g
Pepton……….………………………………..5 g pH= 6.8 ±0.2
Saf su………………………………………..1000cc(L)
Bileşimi:4) Et ekstraktı……………………………………3 g
Pepton…………………………………………5 g pH= 7.3±0.2
Tuz (NaCl)…………………………………… 8g
Saf su………………………………………..1000cc(L)
Hazırlanışı: Bileşenleri tartılır ve 1 L saf suda çözündürülür. pH’ı ayarlanır. Test tüplerine 10–15 ml kadar dağıtılır. Tüpler otoklavda 121 ºC’ta 15 dakika sterilize edilir.

Mac Conkey Agar: 

Koliform grubu bakterileri ile Salmonella ve Shigella bakterilerinin ayrım ve tanımlanmasında kullanılan selektif besi yeridir.
Bileşimi: Pepton……………………………………….20g
Laktoz…………………………………………10 g
Tuz (NaCl)……………………………………5 g pH= 7.1 ±0.2
Bile Salts No.3……………………………….1.5 g
Nötral kırmızısı………………………………0.03 g
Kristal viyole………………………………….0.001 g
Agar………………………………………….13.5 g
Saf su………………………………………..1000cc(L)
Hazırlanışı: Besi yeri bileşenleri tartılır ve 1 L soğuk saf suda çözündürülür. Agar eriyene kadar karıştırılarak kaynar su banyosunda tutulur. pH’sı ayarlanır. Tüplere 15 – 20 ml kadar paylaştırılıp otoklavda 121 ºC’ta 15 dakika sterilize edilir.

Nutrient Agar: 

Agar katılmış Nutrient Broth besi yeridir.
Bileşimi:1. Et ekstraktı……………………………………1 g
Maya ekstraktı………………………………..2 g pH=7.4 ±0.2
Pepton…………………………………………5 g
Tuz (NaCl)……………………………………5 g
Agar………………………………………….15 g
Saf su………………………………………..1000cc(L)
Bileşimi:2. Et ekstraktı……………………………………10 g
Pepton…………………………………………10 g pH 7.5±0.2
Tuz (NaCl)……………………………………5 g
Agar…………………………………………..15 g
Saf su………………………………………..1000cc(L)
Bileşimi:3. Et ekstraktı……………………………………3 g
Pepton……….………………………………..5 g pH 6.8 ±0.2
Agar…………………………………………..15 g
Saf su………………………………………..1000cc(L)
Bileşimi:4. Et ekstraktı……………………………………3 g
Pepton…………………………………………5 g pH 7.3±0.2
Tuz (NaCl)…………………………………… 8g
Agar…………………………………………..15 g
Saf su………………………………………..1000cc(L)
Hazırlanışı: Bileşenleri tartılır ve 1 L saf suda çözündürülür. Agar eriyene kadar kaynar su banyosunda tutulur pH’sı ayarlanır. Otoklavda 121 ºC’ta 15 dakika sterilize edilir.

Brillant Green Bile Broth: 

Koliform grubu bakterilerinin tanımlanması, doğrulanması, izole edilmesi ve sayılmasında kullanılan selektif besi yeridir.
Bileşimi: Pepton………….………………………………10 g
Laktoz………………………………………….10 g pH= 5.6±0.2
Ox-bile(safra tuzları)……………………………20 g
Brillant gren veya %1’lik Brillant gren sulu
çözeltisinden 13.3 ml)…………………………0.0133 g
Saf su………………………………………… 1000cc(L)
Hazırlanışı: Hazır besi yerinden 40 g tartılır. Üzerine 1L saf su eklenip kuvvetlice karıştırılır. Besi yeri hazır değilse pepton ve laktoz 500 ml saf suda çözündürülür. Daha sonra 200 ml saf suda ox-bile çözündürülür. İki çözelti birbirine karıştırılır ve %1’lik Brillant green çözeltisinden 13.3 ml eklenir. Hacim saf su ile litreye tamamlanır pH’sı ayarlanır. Hazırlanan besi yeri, içine ters olarak Durham tüpleri yerleştirilmiş test tüplerine
10’ar ml dağıtılıp, otoklavda 121 ºC’ta 15 dakika sterilize edilir.

Violet Red Bile Agar: 

Koliform grubu bakterilerinin tanımlanması ve sayılmasında kullanılan selektif besi yeridir.
Bileşimi: Maya ekstraktı…………………………………3 g
Pepton………………………………………..7 g
Laktoz…………………………………………10 g
Tuz (NaCl)……………………………………5 g pH=7.4 ±0.2
Bile Salts No.3……………………………….1.5 g
Nötral kırmızısı………………………………0.03 g
Kristal viyole..……………………………….0.002 g
Agar………………………………………….15 g
Saf su………………………………………..1000cc(L)
Hazırlanışı: Hazır karışımdan 41.5 gr tartılır ve 1L saf su eklenip, agar eriyene kadar kaynar su banyosunda tutulur.Su banyosunda kaynama sıcaklığında en çok 5 dakika tutulur. pH’sı ayarlanır. VRBA otoklavda sterilize edilmez. Çünkü VRBA besi yeri ekim yapıldıktan sonra 35–37 ºC’ta 20–24 saat inkübasyon uygulanır. Besi yerinin su banyosunda kaynatılması sırasında ölmeyen mikroorganizmalar olsa bile bu kısa inkübasyon süresinde gelişemezler.

Baird-Parker Agar: 

Gıdalardaki stafilokok bakterilerinin tanımlanma ve sayımlarında kullanılan selektif besi yeridir.
Bileşimi: Triptofan…………………………………..10g
Et ekstraktı………………………………….5 g
Maya ekstraktı………………………………5 g pH=7.0 ±0.2
Sodyum piruvat…………………………… 10 g
Glisin……………………………………….12 g
Lityum klorür………………………………..5 g
Agar…………………………………… ……20 g
Saf su……………………………………….950 ml
Hazırlanışı: Hazır karışımdan 63 g tartılır ve 950 ml saf su ile çözündürülür. Agar eriyene kadar kaynar su banyosunda tutulur. pH’sı ayarlanır. Tüpler otoklavda 121 ºC’ta 15 dakika sterilize edilir. Otoklavlamadan sonra besi yeri sıcaklığı 40-450C’ye düşürülüp aseptik koşullarda 50 ml egg yolk- tellurit emülsiyonu eklenerek kuvvetlice karıştırılır.

Plate Count Agar: 

Gıdalarda, su,süt ve ürünlerinde canlı bakteri sayımlarında kullanılır.
Bileşimi: Tripton…..……………………………………5 g
Maya ekstraktı………………………………..2.5 g pH= 7.0 ±0.2
Glikoz.…………………………………………1 g
Agar……………………………………………15
Saf su………………………………………..1000cc(lt)
Hazırlanışı: Besi yeri bileşenleri tartılır ve 1L saf suda çözündürülür. Agar eriyene kadar kaynar su banyosunda tutulur. pH’sı ayarlanır ve gerekirse erlen, tüp vb dağıtılarak otoklavda 121 ºC’ta 15 dakika sterilize edilir.

Simmons Sitrat Agar: 

Enterobacteriaceae familyası bakterilerinin ayrımları için kullanılan besi yeridir.
Bileşimi: Magnezyum sülfat ……………………………0.2 g
Amonyum di hidrojen fosfat………………….0.2 g
Tuz (NaCl)……………………………………5 g pH=7.1 ±0.2
Sodyum amonyum fosfat……………………. 0.8 g
Tribazik sodyum sitrat…………………………2 g
Brom timol mavisi……………………………..0.08 g
Agar…………………………………………..15 g
Saf su…………………………………………1000cc(L)
Hazırlanışı: Hazır karışımdan 23 g tartılır ve 1L saf su ile çözündürülür. Agar eriyene kadar kaynar su banyosunda tutulur. pH’sı ayarlanır. Test tüplerine 10 – 15 ml kadar dağıtılır. Tüpler otoklavda 121 ºC’ta 15 dakika sterilize edilir. Otoklavlamadan sonra tüpler yatık olarak katılaştırılır.

Laktoz Broth: 

Gıdalarda, su, süt ve ürünlerinde koliform grubu bakterilerinin varlığının saptanması ve doğrulanması amacıyla kullanılan selektif besi yeridir.
Bileşimi:3 Et ekstraktı……………………………………3 g
Pepton……….………………………………..5 g pH=6.7-7.1
Laktoz…………………………………………5 g
Saf su………………………………………..1000cc(lt)
Hazırlanışı: Besi yeri bileşenleri tartılır ve 1 L saf suda çözündürülür. pH’sı ayarlanır. Hazırlanan besi yeri içine ters olarak Durham tüpleri yerleştirilmiş test tüplerine 10’ar ml dağıtılıp otoklavda 121 ºC’ta 15 dakika sterilize edilir.Eosin Metilen Blue Agar: 

Enterobacteriaceae familyası Gram (-) bakterilerinin ayrımı ve izolasyonu için kullanılır.
Bileşimi: Pepton…………………………………….10 g
Laktoz…..…………………………………5g
Sakkaroz…………………………………..5 g
Di potasyum hidrojen fosfat………………2 g pH= 7.2±0.2
Eosin Y……………………………………0.4
Metilen mavisi……………………………0.065 g
Agar……………………………………….13.5 g
Saf su…………………………………… ..1000cc(L)
Hazırlanışı: Hazır karışımdan 36 gr tartılır ve 1 lt saf su eklenip, agar eriyene kadar kaynar su banyosunda tutulur. pH’ı ayarlanır. Otoklavda 121 ºC’ta 15 dakika sterilize edilir. Besi yeri 60 ºC’lara düşürülür. Sterilizasyon sırasında ısı etkisiyle metilen mavisi indirgendiğinden besi yeri turuncu renge dönüşür. Metilen mavisinin okside olup kendi mavi-mor renge dönmesi için besi yeri çalkalanıp, aseptik koşullarda, steril petri kutularına 15 – 20 ml kadar boşaltılır.

‎Laboratory‎ > The Techniques of Pure Cultures

Purpose

     Recognition of media which used  for cultivaring microorganisms and their preparation and sterilization. Learning of to colony selection techniques with pure culture techniques and inoculation of the cultures to different media by transferring techniques.

Theory

      The Techniques of Pure Cultures  

     Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is onlyone microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation. Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are called disinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

Materials

·       Nutrient Agar

·       Nutrient broth

·       Autoclave

·       Beaker

·       Balon Tube

·       Distilled Water

·       Bunson Burner

·       Stirring magnet

·       Cotton

·       Rope

·       Flask

·       Test Tubes

·       Aliminium foil

·       Electrical heater

·       Sterile pipettes

·       Sterile petri dishes

·       Escherichia coli in Nutrient broth

·       Sterile Nutrient agar slant,plate and deep

·       Needle

·       İnoculation loop

·       Alcohol

·       Dilution tubes

Procedure

     Preparation of Nutrient broth: 3.2g of Nutrient broth media was taken and was placed in the beaker.A magnetic stirrer was added.After 400ml of distilled water was added in the beaker.İngredients was mixed.This mixture was distibuted into test tubes about 5-6ml for each tubes.Next, mouth of tubes were closed by

Results and Calculations

Discussion

İn this experiment we learned to prepare different media according to a certain microorganisms and a certain purposes. These media are anaerobic, synthetic, transport, enriched, selective, differential and microbiological assay media. For example; in anaerobic media; oxygen is removed from media with reducing agent, in synthetic media; their chemicals and concentration is known and identified, in transport media; microorganism is transferred from place to other place temporarily, in enriched media; number of scarce microorganisms are increased, but during this process growth of other microorganisms are not prevented, in selective media; growth of a special microorganisms is supplied and growth of other microorganisms are prevented, in differential media; appearance and size of microorganisms are determined with indicator, in microbiological assay media; concentration of some substances are measured. These media are prepared from nutrient broth and nutrient agar that broth represents liquid media and agar represents solid media.

Some media are slant media shape some media also are vertical shape. Slant media provide a large surface area so slant media is convenient for aerobic organisms, in vertical (deep) media, microorganism is cultivated toward the penetration of media and Petri plate also supplies a large surface area and because of this it supplies growth of microorganisms in a short time.

Finally; sterilization of media and equipment were learned during experiment. Moreover, required criteria one by one was told us. For sterilization, applied processes are classified as physical and chemical methods. Physical methods are heat, wet sterilization, tyndallization dry heat sterilization, radiation, freezing and bacteriological filtration. Chemical methods are salt, phenol and phenol derivatives, halogens, and alcohols. Chemical and physical methods and their effects on the microorganisms and equipment were learned and examined.

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; microorganisms were observed, another petri plate had been exposed to cough that a few microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.

Media and Sterilization

BACTERIOLOGICAL MEDIA AND STERILIZATION

The purpose of this experiment is that to prepare media from nutrient agar for cultivating microorganisms. Bacteria have special requirements to grow. In order to see bacterial growth, a medium is needed. A MEDIUM is a nutritional environment for bacteria to grow. There are two primary forms of media: Liquid (Broth) and Solid (Agar). The most common solid medium used to grow bacteria in a microbiology lab is Nutrient Agar. The most common liquid medium used in the lab is Nutrient Broth. AGAR is derived from the extract of seaweed (an alga).Agar contains two main components: agarose and agaropectin. Agar contains solidifying factors and therefore has a gelatin like nature. Some agars are used in cooking and preparation of ice cream. The most familiar food is Jell-O. Nutrients such as peptone, tryptone, soy, and beef extract, salt, calcium, magnesium, water, and manganese are added to the agar or broth providing the bacterium with a proper growing environment.

All bacteriological media must first be sterilized before it can be used. Distilled water is added to agar, heated to a boil then autoclaved. An AUTOCLAVE sterilizes the bacterial growth medium so that a pure culture can be obtained. The autoclave sterilizes the medium by subjected it to a temperature of 121° C for 15 to 20 minutes. It uses steam under pressure to obtain this temperature. This will kill any heat resistant bacteria that have contaminated the medium. Once autoclaved, the agar can then be poured into a PETRI PLATE or test tube. When placed in a test tube, it can either be tilted on a slanted board so that it will solidify at a SLANT or remain upright to solidify into a DEEP. Broths are dissolved with water, added to test tubes, capped then autoclaved.

Culture Media

  • Media must include source of C, N, P, S, 4 of the 6 major nutrients (CHNOPS), as well as micronutrients. These are usually present as trace contaminants in water, on glassware, or in chemicals used to make media.

  • Media can be liquid or solid. Use for different purposes:

    1. Liquid media: easiest to prepare and use. Good for growing quantities of microbes needed for analysis or experiments. Unless inoculated with pure culture, cannot separate different organisms.

    2. Solid media: usually made by adding agar, seaweed extract, to appropriate liquid. 1.5% agar is standard for plates. Agar melts at 80-90 deg. C, will remain liquid until temperature cools to 40-42 deg. C. Very few microbes can degrade agar, so it is normally not a source of C, and acts as inert gelling medium.

  • Synthetic of Defined Media: usually relatively simple media, all components are known. Useful for photoautotrophs, also in some experimental situations where want to select mutants unable to use certain compounds, or for radioisotope labeling. Example: you want to select a microbe that can obtain all its nitrogen from atmospheric N2. You would prepare synthetic medium with sources of C, P, and S, but no N source. Organisms would be unable to grow unless they can fix nitrogen from air.

  • Complex Media: composition of media not completely known. Often made from inexpensive organic materials such as slaughterhouse wastes (tryptic digests called tryptone, trypticase, etc.), soybeans, yeast wastes from brewing (rich source of vitamins), animal blood, etc. All our standard laboratory media in MCB 229 are complex media, such as Tryptone agar, TSA (trypticase soy agar), Nutrient agar, etc.

  • Selective Media: media favors the growth of one or more microbes. Example: bile salts inhibit growth of most gram-positive bacteria and some gram-negative bacteria, but enteric bacteria adapted to life in animal gut can grow well. Include bile salts in some media such as EMB, MacConkey agar (will use later in this course) to select for enteric.

  • Differential Mediamedia allows distinguishing between different bacteria that grow. Ex: MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red. Bacteria that cannot ferment the same sugar will grow but not affect pH, so colonies remain white.

  • Note that it is possible to design a medium that is both selective and differential.

Sterilization of Media and Equipment:

Sterilization denotes the use of physical or chemical agents to eliminate all viable microbes from a material, whereas disinfection generally refers to the use of germicidal chemical agents to destroy the potential infectivity of a material and need not imply elimination of all viable microbes. Sanitizing refers to procedures used to lower the bacterial content of utensils used for food without necessarily sterilizing them. Antisepsis usually refers to the topical application of chemicals to a body surface to kill or inhibit pathogenic microbes.

Physical Agents:

Heat:

Heat is generally preferred for sterilizing materials excepts those that it would damage. The agent penetrates clumps and reaches sites that might be protected from a chemical disinfectant. Fungi, most viruses and vegetative cells of various pathogenic bacteria are sterilized within a few minutes at 50 to 70 °C and the spores of various pathogens at 100 °C. The spore of some saprophytes, however, can survive boiling for hours. Because absolute sterility is essential for culture media and for the instruments used in major surgical procedures, it has become standard practice to sterilize such materials by steam in an autoclave at a temperature of 121 °C (250 °F) for 15 to 20minutes.

In using an autoclave, it is important that flowing steam be allowed to displace the air before building up pressure , for in steam mixed with air, the temperature is determined by the partial pressure of the water vapour. Thus, if air at 1 atm (15 psi) remains in the chamber and steam is added to provide an additional gauge pressure at 1 atm, the average temperature will be only 100° (that of steam at 1 atm). More over, heating will be uneven because the air will tend to remain at the bottom of the chamber.

Pasteurization is now used primarily for milk. It consists of heating at 62 °C for 30 minutes or in “flash” pasteurization, at a higher temperature for a fraction of a minute. The total bacterial count is generally reduced by 97% to 99%. Pasteurization is effective because the common milk borne pathogens (tubercle bacillus, Salmonella, Streptococcus, and Brucella) do not form spores.

Moist heat and Dry heat:

Sterilization by heat involves protein denaturation and the melting of membrane lipids as a consequence of disruption of multiple weak bonds. Among these, hydrogen bonds between a >C=O and an HN< group are more readily broken if they can be replaced with hydrogen bonds. Accordingly, sterilization requires a higher temperature for dry than for wet material. Reliable sterilization of glassware and instruments in a dry oven requires 160 °C for 1 to2 hours. In addition, bacteria and viruses, like isolated enzymes, are more stable in an aqueous medium when the water concentration is reduced by the presence of a high concentration of glycerol or glucose.

The role of water in heat denaturation of proteins is illustrated by the usefulness of steam in pressing woolen fabrics (e.i. in shifting the multiple weak bonds between fibrous molecules of keratin).

Freezing:

When a suspension of bacteria is frozen, the crystallization of the water results in the formation of tiny pockets of concentrated solution of salts, which do not themselves crystallize unless the temperature is lowered below the eutectic point (about -20°C for NaCl); at this temperature, the solution becomes saturated, and the salt also crystallizes. The localized high concentrations of salt, and possibly the ice crystals, damage the bacteria, as shown by their increased sensitivity to lysozyme. Only some of the cells are killed, but repeated cycles of freezing and thawing result in the progressive decrease in the viable count.

Ultraviolet Radiation:

With radiation of decreasing wavelength, the killing of bacteria first becomes appreciable at 330nm and then increases rapidly. The sterilizing effect of sunlight is attributable mainly to its content of UV light (300-400 nm). Most of the UV light approaching the earth from the sun, and all of that shorter than 290 nm, is screened out by the ozone in the outer regions of the atmosphere; otherwise, organisms could not survive on the earth’s surface.

Ionization radiations: 

This mechanism is not convenient for routine laboratory use, but intense source of radioactivity are now being used to sterilize food. Public fear of danger from the irradiation is unwarranted, as the activated mutagenic molecules produced by the irradiation are extremely short-lived.

Mechanical Agents:

Ultrasonic and sonic waves:

In the supersonic (ultrasonic) range, with a frequency of 15,000 to several hundred thousand per second, sound waves denature proteins, disperse a variety of materials, and sterilize and fragment bacteria. The effect has not been of practical value as a means of sterilization, but it is useful for disrupting cells for experimental purposes (sonication).

Filtration:

Bacteria- free filtrates may be obtained by the use of filters with a maximum pore size not exceeding 400nm.This procedure is used for solutions that cannot tolerate sterilization by heat (e.g. sera an media containing proteins or labile metabolites). The early, rather absorptive filters of asbestos or diatomaceous earth were replaced by unglazed porcelain or sintered glass, and these in turn have been replaced by nitrocellulose membrane filters of graded porosity. Membrane filters can also used to recover bacteria quantitatively for chemical and microbiological analysis.

Chemical Agents:

Chemical methods of microbial control: Most of the chemical agents are used for disinfecting and cannot achieve sterility. The term disinfectant is restricted to those that are rapidly bactericidal at low concentrations. The activity of a disinfectant depends upon the pathogen/nature of material being disinfected.

Salts: Pickling in brine or thermal treatment with solid NaCl has been used for many centuries as a means of preserving perishable meats and fish. Bacteria differ widely in susceptibility.

Heavy Metals: The various metallic ions can be arranged in a series of decreasing antibacterial activity. With small inocula, Hg + and Ag +. At the head of the list, are effective at less than 1 ppm because of their high affinity for –SH groups. The antibacterial action of Hg 2+ can be reversed readily by sulfhydryl compounds.

Phenol and Phenol derivatives: Phenol (carbolic acid) has an anaesthetic effect at concentration below 1% but at concentrations above 1% it has significant antibacterial effect. Phenol and its derivatives (phenolics), exert antimicrobial effect by damaging plasma membrane, denaturing proteins and inactivating enzymes. The phenolics contain a molecule of phenol that has been chemically altered to reduce its irritating qualities and increase its antibacterial activity in combination with soap or detergent. They are often used as disinfectants. Cresols are a group of phenolic found in Lysol. Another phenolic derivative a bisphenol -hexachlorophene is ingredient in soaps and lotions used as disinfectants. It is effective against gram positive streptococci, and staphylococci which cause skin infections.

Halogens: Particularly chlorine and iodine are antimicrobial agents effective against all kinds of bacteria, many endospores, fungi, and viruses. Iodine inhibits the microbial protein synthesis by combining with the amino acid tyrosine. The germicidal action of chlorine is due to the formation of hypochlorous acid, which forms when chlorine is mixed with water. A liquid form of compressed chlorine is used for disinfecting drinking water, swimming pools, and sewage. Also hypochlorite solutions (200ppm Cl2) are used to sanitize clean surfaces in the food and the dairy industries and in restaurants.

Alcohols: Effective against bacteria and fungi but not endospores and viruses. Alcohol denatures proteins and dissolves lipid component of cell membranes. Alcohol has the advantage of acting rapidly and evaporating without leaving any residue. Ethanol and isopropanol are the two important alcohols most commonly used.

MATERIALS:

  • 600 mL Nutrient agar

  • Autoclave

  • 600 mL distilled water                                

  • a piece of folio

  • Florence flask                                             

  • a piece of cotton

  • Petri plate

  • Heater

PROCEDURE:

Firstly, required amount of nutrient agar was calculated. This amount was 12gr. for 600 mL, then 12gr. of nutrient agar was weighed and was put in Florence flask. Next, 600 mL of distilled water was added above 12gr. of nutrient agar. Afterwards, solution was heated until it completely dissolved and solution had clear appearance, then the prepared solution in Florence flask was put in the autoclave at 121 °C for 2 hours for sterilisation. Solution was cooled to about 45 °C after than 2 hours. Finally, about 20 mL of solution was poured into each Petri plate and solutions in Petri plate were waited until it solidified.

DISCUSSION:

In this experiment, we prepared Nutrient Agar Media for Petri plate. Previously, a certain amount of nutrient agar was mixed with distilled water and then solution was heated, and it was put in autoclave for sterilization, after then two hours as far as about 45 °C itwas cooled. Afterwards, approximately 32 Petri plates it was poured.

During experiment, we learned to prepare different media according to a certain microorganisms and a certain purposes. These media are anaerobic, synthetic, transport, enriched, selective, differential and microbiological assay media. For example; in anaerobic media; oxygen is removed from media with reducing agent, in synthetic media; their chemicals and concentration is known and identified, in transport media; microorganism is transferred from place to other place temporarily, in enriched media; number of scarce microorganisms are increased, but during this process growth of other microorganisms are not prevented, in selective media; growth of a special microorganisms is supplied and growth of other microorganisms are prevented, in differential media; appearance and size of microorganisms are determined with indicator, in microbiological assay media; concentration of some substances are measured. These media are prepared from nutrient broth and nutrient agar that broth represents liquid media and agar represents solid media.

Some media are slant media shape some media also are vertical shape. Slant media provide a large surface area so slant media is convenient for aerobic organisms, in vertical (deep) media, microorganism is cultivated toward the penetration of media and Petri plate also supplies a large surface area and because of this it supplies growth of microorganisms in a short time.

Finally; sterilization of media and equipment were learned during experiment. Moreover, required criteria one by one was told us. For sterilization, applied processes are classified as physical and chemical methods. Physical methods are heat, wet sterilization, tyndallization dry heat sterilization, radiation, freezing and bacteriological filtration. Chemical methods are salt, phenol and phenol derivatives, halogens, and alcohols. Chemical and physical methods and their effects on the microorganisms and equipment were learned and examined.