1. Classification of bacteria
L. Dalia Kamal Eldien
MSc in Microbiology
Lecture NO: 3

2. Questions for revision the previous lecture
What are the functions of bacterial cell wall?
Name the two sugar types of peptidoglycan
The movement organ in bacterial cell is ……
Name the different flagella arrangement in bacteria
Define the germination process in sporulated bacteria

3. Objectives At the end of this lecture, the student should know:-
The methods of bacterial classification
Emphasis on morphological& staining classification

4. Introduction
In biology, a cell is defined as the fundamental unit of any living organism because, the cell exhibits the basic characteristics of life
Bacteria and blue-green algae are prokaryotes, while fungi, other algae, fungi and parasite are eukaryotes.
Bacteria are prokaryotic microorganisms that do not contain chlorophyll.
They are unicellular and do not show true branching, except in higher bacteria like actinomycetese

5. Mrs.:Dalia Kamal Eldien MSC in microbiology
Classification
The bacteria are classified depending on different methods, include:-
Morphology
Staining reactions
Growth Requirements(Cultural characteristics)
Biochemical reactions
Antigenic structure
Increasingly by their genetic composition using specialized molecular biology techniques.
Mrs.:Dalia Kamal Eldien MSC in microbiology

6. Morphological classification of bacteria
Bacteria are classified according to morphology in to:-
Cocci: (Singular: coccus)
Bacilli rods: (Singular: rod, bacillus)
Cocobacilli: (Singular: cocobacillus)
Vibrios: (Singular: vibrio)
Spiral: (Singular: spirillum)
Spirochetes: (Singular: spirochete)
Mrs.:Dalia Kamal Eldien MSC in microbiology

7. Mrs.:Dalia Kamal Eldien MSC in microbiology
Cocci
These are round or oval bacteria measuring about 0.5–1.0 μm in diameter.
When multiplying, cocci may form pairs, chains, or irregular groups, show characteristic cellular arrangement
Mrs.:Dalia Kamal Eldien MSC in microbiology

8. Mrs.:Dalia Kamal Eldien MSC in microbiology
Cocci
According to the plan of cellular division, cocci may be arranged in
Pairs (diplococci) e.g. meningococci and gonococci.
Chains (streptococci) e.g. Streptococcus pyogenes.
Grape like clusters (staphylococci) e.g. Staphylococcus aureus.
Groups of four (tetrads) or eight (sarcina)
Mrs.:Dalia Kamal Eldien MSC in microbiology

10. Cocci in pairs

11. Cocci in cluster

12. Cocci in chain

13. Cocci in tetrads

14. Cocci in sarcina

15. Basic arrangement of cocci

16. Mrs.:Dalia Kamal Eldien MSC in microbiology
Bacillus
These are stick-like bacteria with rounded, tapered (fusiform), square, or swollen ends.
They measure 1–10 μm in length by 0.3–1.0 μm in width.
Bacilli are arrange in fewer groups, or singly, or in short chain
Mrs.:Dalia Kamal Eldien MSC in microbiology

17. Bacillus
When multiplying, bacterial rods do not usually remain attached to one another, but separate.
Occasionally, however, they may:–
Form chains, e.g. Streptobacillus species.
Form branching chains, e.g. lactobacilli.
Mass together, e.g. Mycobacterium leprae.
Remain attached at various angles resembling Chinese letters, e.g. Corynebacterium diphtheriae.

18. Bacilli arrangement

19. Bacilli in single

20. Bacilli in chain

21. Bacilli in branching chain

22. Bacilli masses together

23. Bacilli resembling Chinese letters

24. Mrs.:Dalia Kamal Eldien MSC in microbiology
Vibrios
These are small slightly curved rod measuring 3–4 μm in length by 0.5 μm in width.
Most vibrios are motile with a single flagellum at one end.
They show a rapid darting motility, e.g. Vibrio cholerae.
Mrs.:Dalia Kamal Eldien MSC in microbiology

25. Vibrio (Helicobacter pylori)

26. Mrs.:Dalia Kamal Eldien MSC in microbiology
Spiral
These are small, regularly coiled, rigid organisms measuring about 3–4 μm in length.
Spirilla are motile with groups of flagella at both ends.
An example of a spirillum is Spirillum minus.
Mrs.:Dalia Kamal Eldien MSC in microbiology

27. Spiral bacteria

28. Mrs.:Dalia Kamal Eldien MSC in microbiology
Spirochetes
These are flexible, coiled, motile organisms.
They progress by rapid body movements.
Most are not easily stained by the Gram method.
Mrs.:Dalia Kamal Eldien MSC in microbiology

29. Spirochetes

30. Identify the following bacteria structure?

32. Bacterial Classification Staining reaction
Classification according to staining reactions depend in different type of stains
The main stain used in bacteriology is Gram stain
The Gram staining method is named after the Danish histologist Hans Christian Gram (1853 – 1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue
Mrs.:Dalia Kamal Eldien MSC in microbiology

33. Structure of bacterial cell wall
The major components of the bacterial cell wall are long glycan strands that are cross-linked by short peptides containing amino acids in both the d- and l-isoform is called peptidoglycan or murine
While all bacterial cell walls contain peptidoglycan, not all cell walls have the same overall structures.

34. Gram-positive cell wall
In the Gram-positive Bacteria:
The Major component of cell wall several layers of peptidoglycan (50-90% of cell wall)
Accessory polymers perpendicular to the peptidoglycan sheets is a group of molecules called teichoic acids which are unique to the Gram-positive cell wall
In the Gram-positive bacteria, the cell wall is thick (15-80 nanometers)

36. Gram-negative bacteria
The cell of Gram-negative bacteria consists of a pair of membranes (cytoplasmic and outer) with a thin, intermediate layer of peptidoglycan (10% of the cell wall)
The outer membrane contains lipopolysaccharide (LPS) , it is a polysaccharide attach to unique lipid molecule, known as lipid A , which is toxic to animals
The inner layer consist of phospholipids
No teichoic acids are found in the cell wall of Gram negative bacteria.

38. Cell wall of Gram positive& negative bacteria

39. Principle of Gram stain
The procedure is based on the ability of microorganisms to retain  color of the stains  used during the  gram stain reaction.
Gram-positive  bacteria  are  not  decolorized by alcohol and will remain as purple.
Gram-negative bacteria are decolorized by the alcohol,  losing  the  color of  the primary stain.
After  decolorization step, a counter stain is used to impart a pink color to the decolorized gram-negative organisms.

40. Gram stain Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram's Iodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone)
Counter stain (safranin or neutral red, dilute carbol fuchsin)

41. Technique of Gram stain
Flood the slide with crystal violet solution for one minute. Wash off briefly with tap water
Flood slide with Gram's Iodine solution, and allow to act (as a mordant) for about one minute. Wash off with tap water.
Flood slide with 95% alcohol for 10 seconds and wash off with tap water. It is most important step
Flood slide with safranin solution and allow to counterstain for 30 seconds. Wash off with tap water
All slides of bacteria must be examined under the oil immersion lens of light microscope

42. Set of Gram stain

43. Gram reaction
Differences in Gram reaction between bacteria is thought to be due to differences in the permeability of the cell wall .
Following staining with a basic dye such as crystal violet and treatment with iodine, the dye–iodine complex is easily removed from the more permeable cell wall of Gram negative bacteria but not from the less permeable cell wall of Gram positive bacteria.
Retention of crystal violet by Gram positive organisms may also be due in part to the more acidic protoplasm of these organisms binding to the basic dye (helped by the iodine).

44. Results of Gram stain
Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), so it stains as violet.  
Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, so it stains as red

45. Classification of bacteria according to Gram stain
According to Gram stain, bacteria classify into large groups:-
Gram-positive bacteria Violet color
Gram-negative bacteria Red color

46. Gram-positive bacteria

47. Gram-negative bacteria

48. Identify ???

49. Different bacteria structure

50. Identify ???

51. Identify ???

52. Sources
District Laboratory Practice in Tropical Countries, Part 2 (page 19-20& 38)
Lippincott's Illustrated Reviews: Microbiology, 3rd Edition (2012)- Richard A. Harvey- Bruce D. Fisher- Richard A. Harvey-(chapter6)