1. Bacterial growth MUDr. Lenka Černohorská, Ph.D.

2. Generation time (GT) GT= duration of the growth cycle = duplication time = time of doubling the number of bacteria GT of bacteria: on average 30 min Escherichia coli under ideal conditions 20 min Mycobacterium tuberculosis approximately 24 hrs

3. Growth cycle of bacteria Bacteria reproduce by binary fission Period I (iniciation): Period I (iniciation): the cell grows, inside it proteins iniciating the next step accumulate Period C (chromosome replication): Period C (chromosome replication): it diverges from one spot in both directions opposite one another Period D (division): Period D (division): supply of macromolecules is created cytoplasmic membrane inserts between the replicated chromosomes and separates them cell wall grows into the cell and forms septum that divides the maternal cell into 2 daughter cells

4. Microbial growth curve - Closed System log number of viable cells 10 8 6 4 2 lag phase logarithmic(exponential)phase stationary phase death phase approximately 24 hrs time

5. Microbial growth curve shows the number of viable cells in the logarithmic scale, depending on the age of culture The result: 10 9 cells/24 hrs in the stationary culture (in which nutrients are consumed and products of metabolism accumulate)

6. Microbial growth curve – II Lag phase: microbes grow, but do not divide Logarithmic phase: cells divide with a constant speed (GT is constant); relation between the number of the living cells and the time is exponential Stationary phase: the number of cells is stable Death phase: sometimes it proceeds according to the exponential curve

7. Division and arrangement of bacteria Cocci, in one plane: Cocci, in one plane: streptococci Cocci, in different planes: Cocci, in different planes: staphylococci Rods : transverse division: transverse division: majority (chain of rods) lengthwise division: lengthwise division: mycobacteria corynebacteria: (palisade arrangement)

8. Continuous culture The culture is continually supplied with nutrients and simultaneously disposed of the products of metabolism as well as the reproduced cells Culture vessels are called fermentors - are used in industry for the production of microbial mass/for the production of various substances (antibiotics etc.) nutrients products cells fermentor

9. Facts and cultivation If the generation time is 30 min, 1 cell produces after 24 hrs theoretically: 2 48 = 2.8×10 14 cells, actually it is by 5 orders less (i.e. around 10 9 cells) 10 9 bacteria - an amount that is visible by the naked eye: It is used in cultivation: we use liquid or solid media, where we can see the typical „changes“ of medium made by various bacteria (time of cultivation depends on GT of bacteria)

10. Liquid medium (broth) Possible changes: becomes cloudy pellicle appears at the top sediment appears at the bottom Various liquid broths: selenite-broth broth… TSB, BHI,VL-broth, selenite-broth, broth…

11. Solid medium (agar) Result: bacteria form colonies, some characteristics of colony: colour shape opacity (translucency) profile odour margine (edge) size consistency surface surroundings (hemolysis)

12. Solid media basic – for most of bacteria (blood agar) selective – selection of 1 bacterium (10% NACL – staphylococci) MIU M diagnostic – some kind of bacteria can typically change the medium (MIU to MIU) L-) selective-diagnostic - mixture of previous (ENDO - selective for G-bacteria, diagnostic L+ or L-)

13. Important solid media MCs Sabourad agarLöwenstein- Jennsen, Levinthal agar for bacterial growth: Blood agar, Chocolate agar,Endo, MCs, XLD, MAL, Slanetz- Bartley, Sabourad agar, Löwenstein- Jennsen, 10% NACL, Levinthal agar, VL agar (VLA - anaerobes)… MHMH for ATB testing - MH, MH with blood MIU for diagnostic procedures - MIU, Hajn… for tranport – Amies, Stuart… for longterm storage of cultures (glycerol serum broth)

14. Important agars EndoXLD, MALSlanetz-Bartley Blood agar Chocolate agar Endo XLD, MAL Slanetz-Bartley Levinthal agar VL agarMH Hajn 10% NACL Levinthal agar VL agar MH Hajn Sabourad agar Löwenstein-Jennsen

15. Inoculation of the agar plate 1 2 3 4 1, 2, 3, 4 – sterile bacterial loops used for the inoculation Isolated clear colonies – these are used for the susceptibility testing, biochemistry identification etc.

16. Blood agar with E. coli after 24 h incubation via 37 ºC

17. Thank you for your attention