Microbiological technique media preparation & sterilization.pptx

Microbiological technique media preparation & sterilization.pptx

1. Microbiological technique: Media preparation & Sterilization Dr Showkat Ahmad Wani

2. Introduction

3. What is a Culture Medium? A culture medium is an artificial food that supplies nutrients for the growth of microorganisms in the lab. It contains: Carbon source (like sugar) Nitrogen source (like peptone or meat extract) Mineral salts (like NaCl) Vitamins and growth factors Water If we want the medium to be solid , we add a jelly-like substance called agar (from seaweed). If we want it liquid , we leave out the agar.

4. 3. Types of Culture Media Type of Medium Purpose Example Simple / Nutrient medium For general bacterial growth Nutrient agar, Nutrient broth Enriched medium For fussy (fastidious) bacteria that need special food Blood agar, Chocolate agar Selective medium Allows some microbes to grow, inhibits others MacConkey agar (for Gram-negative bacteria) Differential medium Shows differences in colonies (like color change) Blood agar, MacConkey agar Transport medium Keeps microbes alive while carrying samples Stuart’s or Amies medium

5. Fussy (Fastidious) Bacteria Some bacteria are fussy eaters — they cannot grow on ordinary food (nutrient agar). They need special nutrients such as blood, serum, or growth factors. These are called fastidious bacteria . Examples: 👉 So, just like some people like simple food (rice and dal), and some like fancy dishes (pizza and ice cream), ordinary bacteria grow on simple media, but fussy bacteria need special, rich media. Bacterium Special Need Medium Used Neisseria gonorrhoeae Needs blood and extra growth factors Chocolate agar Haemophilus influenzae Needs X and V factors from blood Chocolate agar Streptococcus pneumoniae Needs blood Blood agar

6. Steps in Media Preparation Preparing media is one of the most common and important techniques in microbiology labs. Let’s learn the steps: Step 1: Weighing the medium Take the required amount of dehydrated (powdered) medium using a weighing balance. The amount is usually written on the label of the medium bottle (e.g., 28 grams per liter). Step 2: Dissolving Add the medium to distilled water in a flask or bottle. Stir or heat gently until the powder completely dissolves. Step 3: Adjusting pH Microbes grow best at a particular pH , usually around 7.0 (neutral) . Check using pH paper or a pH meter and adjust using dilute acid or alkali.

7. Step 4: Adding agar (if needed) For solid media , add agar powder (about 1.5% w/v). Agar helps form a firm surface for microbial colonies to grow. Step 5: Dispensing Pour the medium into flasks or test tubes . For agar slants, pour small amounts into tubes and place them at a slanting angle before it solidifies. Step 6: Sterilization Before using, the medium must be sterilized to kill all unwanted microbes (see below). Step 7: Cooling and Pouring After sterilization, cool the medium slightly (to about 45–50°C). Pour it into sterile Petri plates or tubes. Allow it to solidify and then store it in a cool place.

8. Sterilization – What and Why Sterilization means killing or removing all forms of microorganisms , including their spores. It is essential because if our media or instruments are contaminated, we cannot get pure results. Think of it like cooking — if your plate is already dirty before you serve food, everything gets spoiled!

9. Methods of Sterilization There are several ways to sterilize, depending on the material: (a) Autoclaving (Steam Sterilization) Most common method in microbiology. Works like a pressure cooker — uses steam under pressure . Temperature: 121°C Pressure: 15 psi Time: 15–20 minutes Destroys all bacteria, spores, and viruses. Used for: culture media, glassware, cotton plugs, gowns, and instruments.

10. (b) Hot Air Oven (Dry Heat Sterilization) Used for materials that can tolerate high dry heat. Temperature: 160–180°C Time: 1.5–2 hours Used for: glass Petri dishes, metal instruments, pipettes, and dry glassware.

11. (c) Filtration Used for heat-sensitive liquids like antibiotics, sera, and vaccines. Uses membrane filters with tiny pores (0.22 µm) that trap microbes but let liquid pass through. The filtrate becomes sterile.

12. (d) Chemical Sterilization Used for surfaces, hands, and instruments that cannot be heated. Common chemicals: alcohol, phenol, formaldehyde, and bleach .

13. (e) UV Sterilization Uses ultraviolet (UV) light to sterilize air and surfaces inside laminar flow cabinets. Kills microbes by damaging their DNA. Does not penetrate deep, so used only for surfaces.

14. Aseptic Technique Even after sterilization, we must prevent new contamination during work. This is called aseptic technique .

15. Rules to follow: ✅ Work near a flame or inside a laminar flow hood. ✅ Sterilize loops and needles by flaming before and after use. ✅ Keep Petri dishes and test tubes closed as much as possible. ✅ Wash hands and wear gloves and a lab coat. ✅ Do not talk or breathe directly over sterile materials.

16. Summary Table Process Purpose Media Preparation Make food for microbes Sterilization Kill all unwanted organisms Aseptic Technique Keep environment and instruments contamination-free

17. Important Notes Always label your media with name, date, and type before storing. If any medium looks cloudy or has growth before use — discard it , it’s contaminated. Never open autoclaved bottles when they are hot — let them cool to avoid accidents. Key Idea “In microbiology, sterilization is the first step toward success — a single germ can ruin the whole experiment!”

18. Thank you