Bacteria Growth Characteristics

1. Bacteria Growth Characteristics (How do I know what this weird looking blob is that’s growing in my petri dish???)

2. Why do I need to know all this? • Recognizing colony growth characteristics and biochemical reactions to various growth media is important in identifying bacteria. • Colonies look different depending on the type of bacteria • The type of media they grow on can also help you tell the difference between types of bacteria • You will need this info for identifying your unknown sample later!!!

3. Colony characteristics • Colony surface • Smooth • Rough • Wrinkled • Glistening • Examine WELL ISOLATED colonies!!

4. Colony characteristics • Colony color • Soluble: Does the color diffuse into the media/agar? • Insoluble: Does the color stay within the colony? • Color: white-white, white-gray, light purple, yellow-brown, etc. • Color description should be EXTREMELY SPECIFIC!

5. Colony characteristics • Consistency • Take an inoculating loop and poke at the colony. Describe it with one of the following: • Mucoid: resembling mucous • Dry: crusty, brittle • Soft: butter-like consistency

6. Colony characteristics • Visual characteristics • Translucent: see-through • Opaque: no light shines through • Iridescent: rainbow colors in reflected light • Dull: no shine to the colony • Shiny: reflects light • Examine colonies with light sources above AND below the plate!!

7. Colony characteristics • Shape of colony (see handout) • Circular • Irregular • Spindle • Filamentous • Spreading

8. Colony characteristics • Height of colony (see handout) • Raised • Flat • Convex • Heaped • Sunken • Note any UNUSUAL characteristics (i.e., fried egg appearance)

9. Media & biochemical reactions • Key terms • Selective media – contains agents that inhibit growth of a microbe or group of microbes, therefore favoring (or selecting for) the growth of others. Important for primary isolation of a specific type of microorganism from a mixed culture. • Differential media – grow several types of microorganisms but are designed to bring out visible differences among them (i.e., colony color/size, media color changes, formation of gas bubbles/precipitates)

10. Media & biochemical reactions • More key terms • Enrichment media/broth – enhances growth of groups of bacteria by providing special substances or growth factors they require (blood, serum, hemoglobin, vitamins, amino acids, etc.) • May cause some colonies to grow larger than others

11. Media & biochemical reactions • More key terms • Fastidious – bacteria that have complex nutritional requirements • Hemolysis – destruction of red blood cells • Coliforms – G(-) bacteria that ferment lactose, i.e., Escherichia coli

12. Media & biochemical reactions • MSA – Mannitol Salt Agar: selective medium for isolation of Staphylococci species. Contains 7.5% NaCl that inhibits growth of most G(-) and G(+) microbes. MSA contains beef extract as a nutrient and phenol red as an indicator. When Staphylococcus aureus is grown, it ferments the sugar mannitol, producing yellow colonies. Those Staphylococcus species that do not ferment mannitol remain as small red colonies and the plate remains pink to red.

13. Media & biochemical reactions • MAC – MacConkey: selective, differentiating medium for isolation of G(-) bacilli and differentiation of lactose fermenters from nonlactose fermenters. IT contains bile salt and crystal violet that inhibit growth of G(+) bacteria. If the microbe can ferment lactose, the colonies appear pink to red. If not, colonies appear clear. Medium contains lactose and neutral red indicator.

14. Media & biochemical reactions • TSA – Trypticase Soy Agar: general purpose medium for isolation of fastidious microbes. Contains nutrients for both G(+) and G(-) bacteria.

15. Media & biochemical reactions • BLD – Sheep Blood Agar: enriched, non-selective medium. It is TSA with 5% sheep red blood cells added to it. G(+) and G(-) bacteria can grow on this medium. Hemolytic patterns of certain bacteria may be seen: • Alpha hemolysis – incomplete hemolysis; medium looks green • Beta hemolysis – total clearing of medium • Gamma hemolysis – nonhemolytic; no change in color of medium

16. Media & biochemical reactions • EMB – Eosin Methylene Blue agar: selective, differential medium for isolation of G(-) bacilli and differentiation of lactose fermenters from nonlactose fermenters. Contains lactose, eosin dye, and methylene blue dye. The dyes are toxic to G(+) bacteria. When bacteria ferment the lactose, the pH decreases and the medium changes to a purple color. G(-) bacteria that ferment lactose have a green metallic sheen. Those that cannot ferment lactose appear clear.

17. Media & biochemical reactions • CNA – Colistin-nalidixic Acid agar: for isolation of G(+) bacteria. Medium contains sheep blood agar and the antibiotics colistin and naladixic acid added. Colistin disrupts the cell membrane and nalidixic acid blocks DNA replication in G(-) microbes.

18. Media & biochemical reactions • PEA – Phenylethyl Alcohol Agar: selective medium that inhibits swarming of Proteus species. G(+) facultative anaerobes such as Staphylococcus, Streptococcus, and Corynebacterium grow on this medium.

19. Media & biochemical reactions • CHOC – Chocolate agar: enriched, nonselective medium to isolate fastidious microbes. Neisseriaspecies and Haemophilus species grow well on it. This medium is heated to destroy RBCs and release NAD to aid in growth of bacteria. It also has IsoVitaleX, which contains dextrose, cysteine, vitamin B12, thiamine, and ferric nitrate.

20. Media & biochemical reactions • TSB – Trypticase Soy Broth: general purpose medium for all types of microorganisms. In a liquid form that is great to use for growth of stock cultures.

21. Media & biochemical reactions • Urea Broth: for identification of urease producers such as Proteus, Morganella, Klebsiella pneumoniae and some Enterobacter species. The enzyme urease splits urea into alkaline end products. This results in an increased pH and a color change in the indicator to pink-red.

22. Media & biochemical reactions • GN Broth: an enrichment broth used to inhibit G(+) microbes. It contains bile salt that is toxic to G(+) bacteria and is inhibitory to normal flora coliforms of the digestive system.