1. Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital, Brno MUDr. Lenka Černohorská, Ph.D. Microbes and their environment Microbes and their environment Lecture for 2nd-year students March 13th, 2010

2. Environmental factors 1.water 2.nutrients 3.temperature 4.osmotic pressure 5.pH 6.redox potential 7.radiation 8.toxic substances

3. 1. Water 80 % of bacterial cell weight. 15 % of spore live weight Hygrophile organisms (most of bacteria): require freely accessible water Resistance to drying Low: gonococci More resistant: skin flora – staphylococci, acidoresistant (mycobacteria) Resistant: actinomycetes, moulds, parasite cysts, helminth eggs Highly resistant: bacterial spores

4. Water availability Degree of water availability = water activity of the environment (a w ) a w of pure water = 1,0 a w is inversely related to osmotic pressure (higher osmotic pressure, the lower a w ) a w levels tolerated by different microbes: G– bacteria a w ≥ 0,95 (meat) G+ bacteria‚ most of yeasts a w ≥ 0,9 (ham) moulds and yeasts a w ≥ 0,6 (chocolate ) Lowering of water activity is useful for food conservation drying – meat, mushrooms, fruit concentration – preparing jam salting – meat, fish, butter adding sugar – syrup, jam, candied fruit

5. 2. Nutrients 2. Nutrients Most microbes do not grow in pure water The problem lies in keeping the water pure In distilled water Pseudomonas aeruginosa multiply In shower outlets: Legionella pneumophila More about nutrients in previous + practical lectures (liquid, solid media…)

6. 3. Temperature Minimum – sometimes <0 °C Optimum – psychrophiles: 0 – 20 °C mesophiles: 20 – 45 °C thermophiles: 45 – 80 °C hyperthermophiles: >80 °C Maximum – sometimes >110 °C Growth temperature range: narrow (gonococci 30 – 38,5 °C) wide (salmonellae 8 – 42 °C)

7. Impact of the temperature on bacterial growth growth optimum growth optimum growth minimum growth maximum growth minimum growth maximum 32 35-37,5 39 upper killing temperature lower killing temperature 6012 °C°C°C°C growth interval bacteria do not grow, but can alive bacteria do not grow, but alive ex. is theoretical

8. Impact of cold Cold shock: gonococci on cold media from refrigerator die Low minimum growth temperature: 5 °C survive yersiniae, listeriae Slow ordinary freezing and repeated thawing damages microbes Lyofilization – dry and freeze quickly – is used for longterm storage

9. Impact of higher temperature Higher temperature → shock and gradual killing of cells. Number of killed cells depends on the duration of higher temperature Relation between the number of surviving cells and the duration of temperature effect is logarithmic one Time needed for killing the whole population depends on the original number of microbes

10. Temperature – important parameters I Relation between the duration of heating and the number of surviving microbes log 10 number of surviving microbes 6 D = decimal reduction time = 5 = time required to reduce the 5 = time required to reduce the 4 number of microbes to 1/10 = 4 number of microbes to 1/10 = 3 = time required to kill 90 % of 3 = time required to kill 90 % of 2 D the microbes 2 D the microbes 1 1 2 3 4 5 6 (min) 1 2 3 4 5 6 (min)

11. Temperature – important parameters II Thermal death point = the lowest temperature at which a microbe is killed in a certain time (cca 10 minutes). It depends on the kind of microbe,on its conditions, number and the effect of environment Thermal death time = the shortest time to kill the given number of microbes. For most bacteria - 10-15 min at 60-65 °C

12. 4. Osmotic pressure (OP) Hypotonicity Hypotonicity – protection by the cell wall Hypertonicity Hypertonicity – mostly harmful (salting meat, candying fruits) Halophiles high OP is tolerated by: Halophiles halotolerant: halotolerant: enterococci, staphylococci obligate: obligate: halophilic vibrios moulds tolerate contents of saccharose (jam)

13. 5. pH Neutrophiles: growth optimum pH 6 - 8 Alcalophiles: Vibrio cholerae (pH 7,4 – 9,6) alcalotolerant: Proteus (splits urea) Enterococcus (large pH range 4,8 – 11) Acidophiles: facultative: yeasts, lactobacilli (>3), coxiellae (low pH in a phagosome) obligate: Thiobacillus thiooxidans (pH <1)

14. 6.Redox potential (rH) depends on the composition of environment and atmosphere Aerobes – Aerobes – rH>200 mV. Strict aerobes 1 require oxygen to grow Anaerobes – Anaerobes in our body: Anaerobes – rH≤0 mV. Strict anaerobes 2 cannot grow in the presence of oxygen. In nature as well as in our body prosper owing to the cooperation with aerobes and facultative anaerobes. Anaerobes in our body: large intestine (99 % of intestinal microorganisms)‚ vagina‚ oral cavity (sulci gingivales) Facultative anaerobes 3 Facultative anaerobes 3 – can grow in both environments Microaerophiles 4 Microaerophiles 4 - grow best at a low oxygen concentration Capnophiles 5 Capnophiles 5 – grow best at CO 2 enriched atmosphere 1 2 3 4 5 1 2 3 4 5 co 2

15. 7. Radiation UV radiation UV radiation (max. at 260 nm) In nature: airborne bacteria are protected by pigments Artificially: is used for surface or air disinfecting and for destroying traces of DNA in PCR laboratories Ionizing radiation Ionizing radiation (X rays‚ gamma rays) For sterilization of disposable syringes, infusion sets, sutures, tissue grafts… !Very resistant: bacterial spores

16. 8. Toxic substances The impact increases with concentration and duration of action Resistance of different types of microbes against various toxic substances differs !!G- bacteria are more resistant than G+ ones t c

17. Sterilization x disinfection Sterilization = removing all microorganisms Sterilization = removing all microorganisms from objects/environment Disinfection = removing agents of infection Disinfection = removing agents of infection from objects/environment/a bodily surface Disinfection interrupts the spread of infection

18. Types of disinfectants 1.Oxidizing agents 2.Halogens 3.Alkylating agents (aldehydes) 4.Cyclic substances (phenols) 5.Acids and alkali 6.Heavy-metal derivatives 7.Alcohols 8.Surface-active agents (QACs) 9.Others (more in practise…)

19. Thank you for your attention