This document discusses various methods used in clinical microbiology for the diagnosis of bacterial infections, including specimen collection, microscopy, and bacterial culture. It emphasizes the importance of proper specimen collection for accurate diagnosis, describing rules for collecting samples from the correct site and in optimal quantities while avoiding contamination. Microscopy techniques described include staining methods like Gram stain, Ziehl-Neelsen stain, and Giemsa stain to identify bacteria, mycobacteria, and parasites under an optical microscope. Bacterial culture methods involve using various liquid and solid growth media suited to different microbial needs to isolate and identify infectious agents.

1. Bacteriologic diagnosis
collection of samples, microscopy,
isolation & identification (culture media)

2. Clinical microbiology
Purposes:
• Etiology of infection
• Most effective antimicrobial agents
• Detection of asymptomatic carriers of microbes
Laboratory methods:
A. Direct – detection of causative (infecting) agent
B. Indirect – detection of infected host response(s) to
infection

3. Specimen collection
• Crucial for confirming a certain microorganism as cause of the
clinically suspected infectious disease
• Improper specimen collection may cause:
– Failure to recover the microorganism (no growth on culture medium)
– Incorrect / harmful therapy e.g directed against a comensal /
contaminant microorganism
E.g. Klebsiella pneumoniae:
- recovered from sputum of pneumonia patient;
- causative agent of pneumonia BUT also may colonize the naso-pharynx
- If sputum sample consisted mostly of saliva then isolating
K.pneumoniae might not reflect the true cause of the patient‘s
pneumonia but saliva contamination of the sputum sample

4. Specimen collection (continued)
Rules for correct specimen collection:
1. Source: actual infection site; minimal contamination
from adjacent tissues, organs, secretions e.g. throat
swabs from peritonsillar fossae and posterior
pharyngeal wall, avoiding contact with other oral areas
2. Optimal moment: depending on the natural history
and pathophysiology of the infectious process e.g.
Typhoid fever: blood – 1st week; feces and urine –
2nd-3rd week
3. Sufficient quantity

5. Specimen collection (continued)
Rules for correct specimen collection (continued):
4. Appropriate collection devices, containers
+ transport systems (container ± transport medium): main objective
to decrease time between collection and inoculation to prevent lack
of recovery of certain bacteria
5. Sample collection before antibiotics (if possible)
6. Smears performed to supplement culture (if possible)
- Assessment of inflammatory nature of specimen → aid the clinical
integration (meaningfulness) of the culture result
- Gram smears e.g. Gram negative bacilli + no growth on aerobic culture
(wrong atmosphere or wrong media i.e. fastidious microbes e.g.
Legionella)

6. Specimen collection (continued)
Rules for correct specimen collection (continued):
7. Labeling of specimen containers & Request form:
• Legible
• Minimun information:
– Patient name; identification number (hospital file, practice log book, etc)
– Source of specimen; clinician + contact data (phone no)
– Date and hour of collection
– Clinical diagnosis (suspected infection)
– Treatments (antibiotics?...)

8. Pre-analytic phase: Transport (continued)
Transport media

9. Triple packaging

10. Specimen receipt & preliminary
observations
• Specially designed area / room for receiving and
recording samples
• Rules for manipulating samples and accompanying
documents (UNIVERSAL PRECAUTIONS):
– Samples: biological safety cabinet (BSC), personal protective
equipment (PPE): lab coat, gloves, eye&respiratory protection
– Documents – handled by different person / at different stage e.g.
either before or after preliminary examination/processing of
sample (after removal of gloves & hand washing) – purpose:
avoid cross contamination of objects (log record book, computer,
pens, etc)

11. Bacterial infections:
direct identification & characterization methods
• Microscopy
• Cultivation
• Antimicrobial sensitivity

12. Microscopy
• Types of microscopes
– Optical - Magnification objectives
• 10x; 40x; 100x for bacteria
– Phase contrast
– Dark field (dark ground)
– Fluorescence – UV light
– Electron

13. Optical microscope

14. Microscopic examination
• Wet mounts (unstained materials)
– Direct light
– Observation of cells (PMN, macrophages),
mobile germs in liquid samples (urine, CSF),
shape and disposition of germs
(cocci/bacilli/spirilli/vibrios)
• Stained smears

15. Microscope glass slide and cover slip

16. Spirochetes – wet mount by dark field
microscopy

17. Treponema denticola – dark field
microscopy + fluorescent dye staining

18. Stained smears
Main steps:
- Smear specimen on microscope glass slide
- Air Drying
- Heat Fixation (flame): help adhesion of specimen to
slide, kill bacteria, favour absorbtion of stain on bacterial
surface
- Staining:
- Monostaining e.g. Methyl blue
- Combined e.g. Gram, Ziehl Nielsen

19. Gram staining
1. heat-fixed smear flooded with crystal violet (primary stain)
2. crystal violet is drained off and washed with distilled water
3. smear covered with ”Gram's iodine” (Lugol) (amordant or helper)
4. iodine washed off: all bacteria appear dark violet or purple
5. slide washed with alcohol (95% ethanol) or an alcohol-acetone
solution (decolorizing agent)
6. alcohol rinsed off with distilled water
7. slide stained with safranin, a basic red dye (counter stain) 2-3
minutes
8. smear washed again, heat dried and examined microscopically
Exact protocol – depending on the kit

20. Gram staining

21. Gram stained smear

22. Streptococcus mutans – Gram stained
smear

23. Ziehl-Neelsen Staining
• used to stain Mycobacterium tuberculosis and
Mycobacterium leprae = acid fast bacilli: stain with carbol
fuschin (red dye) and retain the dye when treated with
acid (due to lipids i.e. mycolic acid in cell wall)
Reagents
• Carbol fuchsin (basic dye) - red
• Mordant (heat)
• 20% sulphuric acid (decolorizer) – acid fast bacilli retain
the basic (red) dye
• Methylene blue (counter stain) – the other elements of
the smear, including the background will be blue

24. Mycobacterium tuberculosis - Ziehl-Neelsen
Staining

25. Giemsa staining
• Smears from blood, vaginal / urethral secretion, bone
marrow aspirate
Steps:
- Fixation with methanol (2-3 min)
- Coloration with Giemsa solution
- Washing – buffered water
- Drying
- Microscopic examination

26. Malaria parasites in blood smear
(Wright/Giemsa staining)

27. Microscopy for various biological specimens
• CSF:
– wet mounts – assess type & no of cells (white/red blood cells)
– Stained smears from centrifugation sediment: Gram, Ziehl-
Neelsen + aditional smear
– Presumptive causative agents:
• High no of PMN on wet mount→ bacterial meningitis Neisseria
meningitidis, Haempohilus influenzae
• Ziehl-Neelsen stained smear – very important in case
M.tuberculosis is suspected (cultures take 2-3 weeks)

28. Microscopy for various biological specimens
• Pus
– Gram stained smears: PMN + staphylococci, streptococci
• Urine
– Gram and Ziehl-Neelsen stained smears prepared from
sediment (after centrifugation of specimen)
– Urinary infection: smear with germs + high no of PMN
• Sputum
– Prewashing of specimen in several, successive Petri dishes (to
remove germs from the pharynx attached to sputum)
– Gram (staphylococci, streptococci), Ziehl-Neelsen
(M.tuberculosis)

29. Cultivation of microbes on culture media

30. General requirements
- acellular, inert media – suitable for most bacteria and
yeasts
- cell cultures / embryonated eggs / animal models –
needed for intracellular microorganisms (ricketsiae,
chlamidiae) and viruses
- Composition of culture media – based upon knowledge
of growth requirements in order to isolate, multiply and
identify bacteria
- Exceptions: bacterial species which cannot be grown on
culture media e.g. Mycobacterium leprae (leprosy),
Treponema pallidum (syphillis)

31. General requirements (II)
• sterility
• nutriets to support microbial growth and multiplication:
– water, carbon, nitrogen, growth factors, vitamins, minerals
• pH: 7.2-7.4 suitable for most germs
– (exceptions: 6.8 for Brucella spp. and 9 for Vibrio cholerae)
• clarity (transparency) →changes induced by bacterial
growth
• aerobiosis / anaerobiosis

32. Composition of culture media
– Peptones = products of animal protein hydrolysis = source of
nitrogen – non standardised composition but suitable for all
cultivable bacteria; included in all commercially available culture
media (Merck, Oxoid, etc)
– Beef extract – obtained by boiling and dehydrating beef =
source of nitrogen (creatine, xantine, uric acid, urea) and carbon
(glycogene, lactic acid)
– Yeast extract – important source of group B vitamins
– NaCl – for adjusting osmolarity (0.9-10%)
– Additional sources of carbon: glycerine, mannitol
– PLUS: solidification agents – agar-agar = gelatin from algae
(nondigestible for bacteria, does not melt at 37°C)

33. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

34. Classification of culture media
(continued)
I. Depending on state of matter:
A. Liquid media
1. Broth
2. Peptoned water
B. Semisolid & solid (gelified with 5% agar)

35. Classification of culture media
(continued)
A. Liquid media:
1. Nutrient broth = powdered beef extract (peptone
content) dissolved in water – commercially available;
used to be prepared by actually boiling beef/horse
meat
- Widely used in microbiology laboratories:
- hemoculture – blood innoculated in liquid media
- identification of isolated bacterial strains by
biochemical tests (e.g.fermentation of sugars)

36. Nutrient broth in test tubes

37. Classification of culture media
(continued)
B. Solid media
- Obtained from liquid media by adding
agar-agar (gelification)
- 1st reported use: Robert Koch 1882 –
cultivation of M. tuberculosis
- Initially gelatin was used - disadvantages:
- Digested by some bacteria
- Liquifies at 37°C – most frequently used
incubation temperature

38. 1882: Fanny Hesse – idea to use agar as
solidification agent instead of gelatin

39. Classification of culture media
(continued)
B. Solid media – Agar (continued)
1000 ml nutrient broth + 25-30 g agar-agar →melted by
boiling + pH adjustment (7.2-7.4)
Features:
- odourless, colourless, nontoxic for microbes
- Nonsoluble in cold water, soluble in boiling water; upon
cooling causes gelification

40. Classification of culture media
(continued)
B. Solid media – Agar (continued)
Advantages:
- Isolated colonies (resulting by multiplication of a single
microbe) → pure cultures can be obtained
- Morphology of bacterial colonies: shape, size, changes
induced in the medium e.g. hemolysis, colour changes,
etc.
- Counting microbes in a biological sample e.g. urinary
infections

41. E.coli colonies on agar

42. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

43. Classification of culture media
(continued)
II. Depending on complexity:
1. Simple media (previously described)
2. Enriched media: blood and other special nutrients may
be added to simple media to encourage the growth of
fastidious microbes e.g. blood agar, ”chocolate” agar

44. Classification of culture media
(continued)
II.2. Enriched media: Blood agar:
- 5-10% mammalian blood (sheep / horse)
- Double purpose:
- Isolation of fastidious organisms and
- Detection of hemolytic activity:
- β-hemolysis - lysis and complete digestion of red blood cell
contents surrounding colony e.g. Streptococcus
haemolyticus
- α-hemolysis - partial lysis – incomplete hemoglobin
digestion → green or brown (due to the conversion
hemoglobin to methemoglobin) e.g. Streptococcus viridans
- γ-hemolysis (or non-hemolytic) - lack of hemolytic activity

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

46. Porphyromonas gingivalis on blood agar
- Involved in periodontal disease and tooth decay (bacterial
plaque)

47. Classification of culture media (continued)
II.2. Enriched media (continued): Chocolate agar
- variant of blood agar in which red blood cells have been
lysed by slow, gradual heating to 80°C in order to
provide additional growth factors contained in red blood
cells
- !Does not contain chocolate!! The name is suggestive for
the brownish colour resulted after red blood cell lysis
- used for growing fastidious respiratory bacteria e.g.
Haemophilus influenzaze, Neisseria meningitidis

48. Attention!
Enriched media are non-selective – i.e.
they contain additional substances aiming
to a better growth & multiplication
≠
Enrichment media are selective i.e.
content is adjusted to favour certain germs
and inhibit others (see below)

49. Left: enriched, nonselective; Right: enrichment, selective

50. Classification of culture media
Main classification criteria:
I. Sate of matter
II. Complexity
III. Purpose

51. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

52. Classification of culture media (continued)
III.1. Selective & enrichment media
- Favour the growth and multiplication of certain bacteria
while suppresing other species
- Very useful for polymicrobial biological products when
attempting to isolate pure cultures
- Used for inoculation of biological products (primary
isolation)
- Composition & cultivation conditions (temperature,
aero/anaerobiosis, etc) adjusted according to the known
growth characters & requirements of the suspected
microbe

53. Classification of culture media (continued)
III.1. Selective & enrichment media (continued)
Liquid selective media and/or cultivation condition –
examples:
- Nutrient broth + acid sodium selenite – Salmonella spp
- Peptone water – Vibrio cholerae – the alkaline pH (9)
inhibits other species
- Temperature: +4°C – inhibits the growth of most bacteria
EXCEPT Listeria spp

54. Classification of culture media (continued)
III.1. Selective & enrichment media (continued)
Solid selective media – same principles, same inhibition
criteria
Chemical inhibitors: antibiotics (chosen depending on the
known natural sensitivity of bacteria) e.g. Vancomycin
added when trying to isolate gram negative anaerobic
bacteria (gram positive anaerobic bacteria are
vancomycin sensitive and their growth will be inhibited)

55. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

56. Classification of culture media (continued)
III.2. Diagnostic media
- Contain indicator systems demonstrating metabolic
characters of certain microbial species (fermentation of
sugars, production of H2S, etc)
E.g. Fermentation of sugars:
nutrients + sugar + pH indicator – in case fermentation
occurs the colour will change indicating the presence of
a bacteria which ferments that particular sugar
- Identification relies on performing a number of tests and
analyzing the ”profile” which is further matched to known
metabolic & growth characters of bacteria

57. III.2. Diagnostic media (continued)
Mannitol Salt Agar (Chapman) - selective medium with a
high salt concentration for the isolation, growth and
enumeration of Staphylococcus species: organisms that
use mannitol turn the medium colour to yellow

58. Chapman agar – mannitol acidification

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

60. Staphylococcus spp – biochemical tests

61. Streptococcus spp – biochemical tests

62. Classification of culture media (continued)
III. Depending on purpose:
1. Selective & enrichment media
2. Diagnostic media
3. Special media

63. Classification of culture media (continued)
III.3. Special media
- Specially designed for certain species
E.g.
- Lowenstein-Jensen for M. tuberculosis
- Tynsdale for C. diphtheriae
- Bordet-Gengou for Bordetella pertussis

64. What if bacteria do not grow?
Troubleshooting
• Wrong culture medium
• Wrong quantities of ingredients
• Wrong pH
• Contamination (improper cleansing and/or sterilization of
plates, tubes, flasks)
• Impaired incubation conditions (power failures during
overnight incubation)
• Improper sample collection/transportation
• Lack of proper quality control of culture media (reference
strains)