This document discusses bacterial culture techniques for isolating and identifying bacteria from clinical samples. Pure bacterial colonies are necessary to determine their characteristics and properties. Bacteria are streaked in patterns on agar plates to separate them into isolated colonies. The colonies are then analyzed based on morphology, including size, shape, color, and other traits. Hemolysis patterns and reactions in biochemical tests of the colonies provide information to identify the bacterial species. Proper culture methods allow clear observation of colony traits for bacterial identification.

1. Bacterial cultures: identification of germs
by morphology of colonies
http://www.slideshare.net/DanaSinzianaBreharCi/bacterial-cu

2. Bacterial colonies
• Isolation of bacterial colonies - essential to
work with pure cultures
• Why?: to determine
– colonial characteristics,
– biochemical properties, and
– other details

3. Bacterial culture
• Selection of primary media depends on
the suspected causative bacteria from
clinical sample.
• Primary inoculation - loop / swab
• Bacterial streaking to obtain isolated
colonies

4. Bacterial culture streaking
• Purpose: to identify and isolate pure
bacterial colonies from a mixed population
i.e. biological specimen
– spread specimen across agar plate
– incubate at a certain temperature for a period
of time

5. Plating
• Liquid specimens - 1-2 drops
• Feces or sputum - dip swab into specimen
• Swabs - direct plating

6. Bacterial culture streaking
• dilute large concentration of bacteria to a smaller
concentration →
• decrease density of bacteria →
• colonies spread apart →
• separation of different types of microbes
• Tools: sterile swab / inoculation loop
• Aseptic techniques

7. Bacterial culture streaking:
”T-Streak” method
• Inoculation loop sterilized (flame) & cooled
• Loop dipped into biological specimen / microbial culture
broth (inoculum – contains many bacterial species)
• Loop dragged across agar surface (back and forth,
zigzag upon 30% of plate)
• Loop re-sterilized (flame) & cooled
• Plate turned 90 degrees
• Loop dragged from previously streaked section (back
and forth, zigzag)
• Repeat last 3 steps once more upon non-streaked plate
area

8. What happens?
Each time the loop gathers fewer bacteria until it
gathers single bacterial cells →colonies:
– 1st plate section: heavy growth – no isolated
colonies
– 2nd plate section: less growth & some
isolated colonies
– 3rd plate section: lowest growth & many
isolated colonies

10. Inoculation of media – general principles
• Order of inoculation: least → most selective
(avoid inhibition of microbes by the selection
agent e.g. salt, antibiotics)
E.g.
1. Agar / blood agar – staphylococci, streptococci
2. Salt agar (Chapman) – S. aureus
3. Vancomycin containing medium – Vancomycin
resistant Enterococcus

11. Blood agar plates
Left: Staphylococcus; Right: Streptococcus

12. Mannitol Salt Agar (Chapman)
- high salt concentration supports growth
of Staphylococcus / inhibits Streptococcus
- mannitol acidification - turn the medium colour to yellow

13. Chapman agar – mannitol acidification

14. Staph. aureus - mannitol fermentation (left side, left plate)
Staph.epidermidis - no mannitol fermentation (right side, left plate)
Streptococcus pneumoniae – plate on the right

15. Blood agar: Enterococcus fecalis (non-hemolytic)
and Streptococcus pyogenes (hemolytic)

16. Enterococcus fecalis on blood agar

17. Vancomycin Resistant Enterococcus fecalis (VRE)
on selective medium containing vancomycin

18. Colonial Characters on Solid Media
• Size
• Shape
• Margins
• Transparency
• Relief
• Type S / R* - see next slide
• Further description
• Colour
• Consistency
• Adherence to medium
• Changes induced in the medium

19. Colony Type: S(smooth)/R (rough)
• type S – smooth, shiny surface, well designed margins,
creamy consistence; homogeneous suspension in saline
solution
• type R - rough, flat, irregular surface and margins, dry,
adherent to the culture medium; granular suspension in
saline solution
• Most pathogens from clinical specimens generate
type S colonies on solid culture media
• Exceptions: C.diphteriae, M.tuberculosis, B.anthracis –
pathogenic strains from clinical samples generate type R
colonies

20. Colonial characters: Colony Size
• Large: Staphylococcus
• Small: Shigella, Salmonella
• Pinpoint (”powder grains”): Streptococcus
• Very small: Neisseria

21. Left: Staphylococcus (large colonies);
Right: Streptococcus (small, powder-like colonies)

22. Left: polymicrobial growth, various colony sizes;
Right: (very small) Neisseria colonies

23. Colonial characters: Colony shape
• Round (Staphylococcus, Streptococcus,
Neisseria)

24. ”Daisy flower” – Corynebacterium

25. Klebsiella pneumoniae: Mucous colonies

26. Colour of colonies (bacterial pigment)
• White / colourless: Staphylococcus epidermidis
• Golden: Staphylococcus aureus
• Green: Pseudomonas aeruginosa

28. ”Golden” colonies: Staphylococcus aureus

29. Green colonies: Pseudomonas aeruginosa

30. Changes induced in medium components
Hemolysis:
- β-hemolysis - complete digestion of red
blood cell contents surrounding colony e.g.
Streptococcus haemolyticus
- α-hemolysis - partial lysis – incomplete
hemoglobin digestion → green or brown
(conversion of hemoglobin to methemoglobin)
e.g. Streptococcus viridans
- γ-hemolysis (or non-hemolytic) - lack of
hemolytic activity

31. Bacterial colonies:
Types of hemolytic activity

32. Alpha-hemolysis: Streptococcus
pneumoniae

33. Beta-hemolysis: Streptococcus pyogenes

34. Gamma hemolysis = No hemolysis:
Left: Enterobacter; Right: Moraxella catarrhalis

35. Changes induced in medium components
• Hemolysis (already discussed)
+
• Fermentation of sugars (lactose, manitol)
• Use of citrate, malonate as sources of carbon
• Production of H2S
Used for identification of bacteria upon
biochemical characters