Practical No 2 Culture Media and Laboratory Equipments

Practical No. 2 Culture Media and Laboratory Equipments Department of Microbiology College of Medicine

General Procedure of Bacteriological Diagnosis Specimens collection General rules The specimens should be representative of the infectious process; Sufficient material; Avoid the contamination of specimens; Be sent to the lab immediately in an appropriate method and examined ASAP. Be collected before using antimicrobial agents, e. g. antibiotics. 2

General Procedure of Bacteriological Diagnosis specimens Morphologic identification microscopy & staining Isolation, identification Biochemical tests Antibiotic sensitivity test 3

Requirements for bacterial growth n Nutrients H 2 O, C-source, N-source, proteins , sugars, Inorganic salts, § Temperature § p. H § humidity culture medium Nutrients, p. H incubator : 37 c, 24 hr 4

The media are contained either in sterile test tubes, plates (Petri Dishes), flasks or screw capped bottles. The media sterilized by moist heat. Sterile Petri dish Sterile screw capped

Basic Ingredient For Culture Media 1 - Peptone: source of protein. 2 - Meat extract: source for N 2, it obtained by: Lean meat +H 2 O Over 24 h cold meat extracts. 3 - Nacl: for isotonic environment. 4 - H 2 O: for humidity. 5 - Agar: it provides no nutritional benefits but used for solidifying purposes, it is obtained from sea weeds, melting point is 92 -95 C°, solidifying point 42 -45 C°, conc. (1. 5 -2 %). 6 - p. H: most pathogenic bacteria needs a PH of 7. 27. 4, this obtained by addition of Nao. H or HCL.

Culture mediumn Is the mixture of various nutrients that is suitable for the ◦ growth of microorganisms. n Types of Culture Media based on the function and chemical components based on the physical state 7

Based on the chemical components: Synthetic medium: prepared manually , known concentration , used for physiological studies. Semi Sythetic medium: prepared manually with known concentration and supplemented , used for isolation and physiological studies. Natural Medium: yeast extract , meat extract their concentration s are not known, used for growth. 8

Based on function: 1 - Isolation medium : All nutrients used for growth of unknown bacteria. e. g. nutrients agar 2 -Enriched Medium Additional or special nutrients (e. g. , serum, growth factors, trace elements) are added to support some fastidious bacterial growth. e. g. blood agar. 9

3 -Selective Medium the medium that can prevent the certain bacterial growth while permitting others. e. g. SS agar 10

Sterile Mac. Conkeys Agar Selective medium Cultured Mac. Conkeys Agar

4 -Differential Medium Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium. e. g. EMB agar (Eosin-methylene blue agar). 12

Because it contains lactose which could differentiate between g-ve bacteria as two group lactose fermenter and non fermenter

5 -Biochemical test: different compounde. e. g. fermentation test, media. Triple sugar iron slant 14

6 - maintenance media : preservation of bacteria for long period of time. 7 -Antibiotic sensitivity test medium: Allows the growth of most bacteria. e. g: Müeller-Hinton agar is most frequently used in this antibiotic susceptibility test. 15

According to physical properties, media can be classified three types: 1. solid medium ( 2 % of agar) agar plate /slant medium 2. semi-solid medium (1 % of agar) 3. liquid medium 1 1 2 3

Liquid (broth) media for primary cultivation Disadvantage? is the inability to obtain colony morphology e. g. , nutrient broth, peptone water, brain heart infusion. Solid media for identification of colony morphology conc. of agar is 2% nutrient agar, Mac. Conkeys agar, blood agar Semisolid media to study motility Contain 1 % of agar

In this exercise, we will prepare a basic nutrient broth medium and also a nutrient agar from commercially available dehydrated stock mixtures containing all necessary ingredients except water. The term ''nutrient broth'' (or agar) refers specifically to basal media prepared from meat extracts, with a few other basic ingredients, but lacking special enrichment. We will also see how liquid and agar media are appropriately dispensed in flasks, bottles, or tubes for sterilization prior to use, and how a sterile agar medium is then poured aseptically into Petri dishes.

Materials: Ø Dehydrated nutrient agar Ø Dehydrated nutrient broth ØA balance, and weighing filter papers ØA 1 -liter Erlenmeyer flask, cotton plugged ØA 1 -liter glass beaker ØA 1 -liter graduated cylinder Ø Glass stirring rode (at least 10 cm long) Ø 10 -ml pipettes (cotton plugged) Ø Test tubes (screw capped or cotton plugged) Ø Petri dishes

Procedures 1 -Read the label on a bottle of dehydrated nutrient agar. It specifies the amount of the content required to 1 liter (100 ml) of medium. Calculate the amount needed for 1/2 liter and weigh out this quantity. 2 - Place the weighed, dehydrated agar in an Erlenmeyer flask. Add 500 ml of distilled water and mix, using a glass stirring rod to prevent lumping. 3 - Set the flask on a tripod, over an asbestos mat. Using a Bunsen flame, bring the rehydrated agar to the boiling degree, slowly stir often. 4 - When the agar mixture is completely dissolved, remove the flask from the flame, close it with the cotton plug, and give it to the instructor to be sterilized in the autoclave (an equipment used for sterilization, will be studied later). 5 - While the agar flask is being sterilized, prepare 500 ml nutrient broth, placing the weighed dehydrate in a beaker for reconstitution and dissolution in water.

6 - Bring the reconstituted broth to a boil, slowly. When fully dissolved, remove from flame and allow to cool a bit. 7 - Using a pipette, dispense 5 -ml aliquots of the broth into test tubes (plugged or capped). The instructor will collect the tubes and sterilize them. 8 - When the flask of sterilized agar is returned to you, allow it to cool to about 50°С (the agar should be warm and melted, but not too hot to handle in its flask). Remove the plug with the little finger of your right hand (which should continue to hold the plug until you are sure it won't have to be returned to the flask), flame the open mouth of the flask, and quickly pour the melted, sterile agar into a series of Petri dishes. The Petri dish tops are lifted with the left hand, and the bottoms are filled to about one-third capacity with melted agar. Replace each Petri dish top as the plate is poured. When the plates are cool (agar solidified), invert them to prevent condensing moisture from accumulating on the agar surface. 9 - Place inverted agar plates and tubes of sterilized nutrient broth (cooled after their return to you) in the 35°С incubator. They should be incubated for at least 24 hours to ensure their sterility before you use them in the next exercise. 10 - Read the results.

Instruments and equipments usually used in bacteriology laboratories; 1. Microscope (with at least 1000 X) 2. Incubator 3. Hot air oven 4. Autoclave 5. Anaerobic jar 6. Microflow (laminar flow) 7. Fume hood 8. Hot plate with magnetic stirrer 9. Electronic Balance 10. Top plate balance 11. Shaking Incubator 12. Shaking water bath 13. Bunsen burner 14. Wire loop & wire needle

Thank You