chapter 6: measuring microbial growth microbial growth requirements

binary fission & biotic potential

microbial population growth

direct methods: dilution & plating

The spread plate method Inoculate plate containing solid medium.

100 μl Bacterial dilution Spread inoculum over surface evenly.

Colonies grow only on surface of medium.

direct methods: microscopic counts

indirect methods: most probable number

indirect methods: spectrophotometry 1 0,8 0,6 0,4 0,2 0

pH optimum

growth requirements: temperature

growth requirements: temperature

food spoilage

growth requirements: osmotic pressure osmoprotectants/compatible solutes

growth requirements: pH All environments • DNA/RNA PO 4 buffer • low pH causes tumbling • membrane gathers H + /OH • ion circuits: Na + /H + antiport • surface proteins pH stable Alkaline environments • Na + /H + antiport • H + motive force changes • Acidic environments not permeable to H +

living with oxygen: anti-oxygen enzymes

oxygen requirements

chemical growth requirements biological macromolecule synthesis

media: meeting physical & chemical needs

MLS-1 growth optima

2,2 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0 DSIC-II (NH4Cl) - 85 hours DSIC-II (glutamine) - 30 hours 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408

time (minutes)

media

DSIC-II Chemically Defined Medium

80g NaCl (sodium chloride) 0.5g NH 4 Cl (ammonium chloride) OR 14mM glutamine 0.6g KH 2.5g K 2 0.1g Na 2 PO SO 2 S 4 2 O 4 3 (monopotassium phosphate) (potassium sulfate)  5H 2 O (sodium thiosulfate)

EM-II Chemically Defined Medium

70g NaCl 0.8g NH 4 Cl 0.8g KH 2 PO 4 10g Na 2 SO 4 0.8g Na 2 S  9H 2 O (Sodium sulfide) dissolved in 10ml H 2 O, filter sterilized & added to cooled media 1ml vitamin solution (10mg biotin, 35mg nicotinamide, 30mg thiamine dichloride, 10mg pyridoxyl chloride, 10mg Ca-panthenoate, 5mg vitamin B 12 , in 100ml dH 2 O) No vitamin solution 1ml trace elements (5.2g EDTA, 190mg CoCl 2  2H 2 O, 0.1g MnCl 2  4H 2 O, 1.5g FeCl 2  4H 2 O, 6mg H 3 BO 3 , 17mg CuCl 2  2H 2 O, 188mg Na 2 MoO 4  2H 2 O, 25mg NiCl 2  6H 2 O, 70mg ZnCl 2 , 30mg VOSO 4  2H 2 O, 2mg Na 2 WO 4  2H 2 O, 2mg NaHSeO 3 , in 1l dH 2 O) 1ml SLA trace elements (1.8g FeCl 2  4H 2 O,; 250mg CoCl 2  6H 2 O,; 10mg NiCl 2  6H 2 O,; 10mg CuCl 2  5H 2 0,; 70mg MnCl 2  4H 2 O,; 100mg ZnCl 2 ,; 500mg H 3 BO 3 ,; 30mg Na 2 MoO 4  2H 2 O; 10mg Na 2 SeO 3  5H 2 O; 1l dH 2 O) 0.2g MgSO 4  7H 2 O (autoclaved separately) 0.2g CaCl 2  2H 2 O (autoclaved separately) 20g NaHCO 3 (autoclaved separately) dH 2 O up to 1L 0.1g MgCl 2  6H 2 O (autoclaved separately) 50mg CaCl 2  2H 2 O (autoclaved separately) 20g NaHCO dH 2 3 (autoclaved separately) O up to 1L, store anaerobically

special media types differential & selective media

Chapter Six Learning Objectives 1.

How do most bacterial cells reproduce? Why do bacterial cells have tremendous biotic potential?

2. Discuss what is happening to a bacterial culture during the fourth phases of population growth. Are they growing, dividing, what is driving the change in population number?

http://professorcrista.com/files/animations/posted_animations/bacterial_growth_curve.html

http://professorcrista.com/files/animations/posted_animations/binary_fission.html

https://www.youtube.com/watch?v=lpI4WCM_9pM&feature=youtu.be

(Go to 4:00) 3. Categorize, discuss the pros and cons and understand the mechanism of action for each of the following methods of counting the number of bacterial cells in culture: serial dilution and spread plating, microscopic counts, MPN, and spectrophotometry.

http://education.wichita.edu/saltymicro/ecology_interactives/serial_dilution.html

4. How do temperature, osmotic pressure, pH and oxygen changes affect the growth of a bacterial culture? How are organisms classified according to their needs in regard to these physical aspects of the environment? What adaptations might various bacterial species have in order to combat less than desirable environments?

5. Six chemical growth requirements were discussed in lecture. What are these used for in the bacterial cell? How does the availability of these affect the growth of a bacterial culture?

6. How are differential and selective media useful in isolating, identifying and enriching for particular bacterial species?

7. How do complex and chemically defined media differ? When is each useful for the routine culture of bacterial cultures?

chapter seven: control of microbial growth

the control of microbial growth

treatment effectivity • • • • • number of microbes/length of exposure microbial characteristics environment: moisture & temperature organic matter vegetations/biofilms 1. cells populate substrate 2. extracellular polymeric substances (EPS) produced & attaches 3 & 4. biofilm architecture develops and matures 5. cells are released from the biofilm

the benefits of a biofilm protection from Abx, toxins and immune cells tobramycin ciprofloxacin  planktonic cells  biofilm cells  biofilm colony open symbols are untreated (control)

terminology • decimal reduction time (DRT)- opposite of decimate • thermal death point (TDP) – lowest temperature when all cells killed in 10 min.

• thermal death time (TDT) – time to kill all cells at given T

heat • dry heat- oxidation – flaming – incineration – hot-air sterilization • moist heat- denaturization – autoclave – pasteurization • not sterile • 63°C for 30 min • flash (UHST): 72°C for 15 sec • ultra heat treatment (UHT): >135°C for <1 sec • moist vs. dry – hot air: 170˚C, 2 hr – autoclave: 121˚C, 15 min

• • filtration physical removal of organisms protects heat-labile components

ionizing & nonionizing radiation ionizing radiation ionizes H 2 O  OH  damages DNA OH  nonionizing radiation damages DNA

chemical control: use-dilution test useful for testing bactericidal properties dip metal ring in bacteria dry at 37°C place ring in disinfectant 10 minutes @ 20°C culture in broth check survivability

chemical control: disk-diffusion method filter paper is soaked with disinfectant paper placed on “seeded” agar plate incubated examined for zone of inhibition

chemical microbial control *

for study

Chapter Seven Learning Objectives 1.

Define sterilization, commercial sterilization, disinfection, antisepsis, degerming and sanitization. Understand what is meant by “-stat,” “-lytic” and “-cide”. http://professorcrista.com/files/animations/posted_animations/chemotherapeutic_agents.html

2. How do the following affect the effectivity of a given microbial control agent: microbial load, exposure length, microbial characteristics, moisture, temperature, organic matter and vegetations/biofilms?

3. How is a biofilm produced? How are the microbes within it so protected from the environment?

4. Why is moist heat better than dry heat at killing microbes? 5. Define thermal death point, thermal death time, and decimal reduction time. 6. When is filtration a useful method of controlling microbial growth?

7. How do ionizing and non-ionizing radiation work to control microbial growth?

8. What experimental method discussed in class is useful to determine the bactericidal properties of a given chemical? Which would you use if you were only concerned with the bacteriostatic properties?

9. How would you rate the various kinds of microorganisms in terms of their resistance to the chemical control of microbial growth?

chapter eight: microbial genetics

the hereditary material Griffith 1927 & Avery, et al. 1944 the “transforming principle” coined by Griffith, identified by Avery

the hereditary material Hershey Chase, 1952

the bacterial chromosome

plasmids • F factor (conjugative plasmid) – genes for sex pili and plasmid transfer • dissimilation plasmids – enzymes to catabolize unusual compounds • R factors – antibiotic resistance