1. Revision
Term One

2. QA Systems
Quality Assurance measures apply to the laboratory analytical work overall, which includes;
identifying the person having the overall responsibility for quality
having laboratory equipment calibrated to recognised standards
>Using only QA.ed materials
Using reference materials +ve and -ve
Validating analyses
Internal proficiency testing.
Joining proficiency testing schemes with other laboratories doing similar tests
>Using correct SOP for each method/activity
Using correct record sheets
Traceability of changes to SOPs and records 2

3. QC systems reagent blanks; verified standard solutions; blind samples
Quality control measures apply to each analytical
test in the laboratory by
use of:
Control chart
reagent blanks;
verified standard solutions;
check samples (from both within the lab and from outside);
blind samples
replicate analyses;
and control charts
Positive and negative controls
All materials QC ed. 3

4. Keeping track of the samples
Lab. No. F7-002
Sample registration gives each sample a unique lab number.
The sample register records all the information about the sample.
Just like a sample’s passport, you should not confuse any sample with any other.
The history of the sample should be traceable throughout.
Sample integrity
Samples recorded on receipt 1 4

5. Beta ( β ) hemolysis (blood agar around bacterial colonies is completely clear, indicating complete breakdown and consumption of hemoglobin)

6. Alpha (α) hemolysis (blood agar around bacterial colonies looks greenish-brown, indicating partial breakdown and consumption of hemoglobin)
Mixed culture from a throat swab; note several β-hemolytic colonies

7. Optochin (Taxo P) sensitivity
- Results with no zone of inhibition
+ results with a zone of inhibition

8. P

9. Catalase Test - +

11. Chapter 21: Diseases of Skin and Eyes 1. Staphylococcal Infections
Gram-positive cocci in irregular clusters
Coagulase negative strains make up to 90% of skin microbiota (S. epidermidis). Only pathogenic when skin is broken or through invasive entry.
Coagulase positive strains tend to be pathogenic. Almost all pathogenic S. aureus strains make coagulase. High correlation between ability to produce coagulase and production of damaging toxins:
Leukocidin: Destroys phagocytic white blood cells.
Exfoliative toxin: Responsible for scalded skin syndrome.
Enterotoxins: Affect gastrointestinal tract.
S. aureus is commonly found in nasal passages. 1 1 1 11

12. 1. Staphylococcal Infections (Continued)
Common staphylococcal diseases
A. Folliculitis: Infection of hair follicles (pimples).
B. Sty: Infected eyelash follicle.
C. Boil (Abscess or Furuncle): More serious infection of hair follicle in which pus is surrounded by inflamed tissue. Usually painful and firm.
D. Carbuncle: Aggregate of several infected follicles (boils). May cause fever, chills, malaise, and death if not treated.
Forms a round, hard, deep area of inflammation, typically on neck and back.
Damages surrounding tissue, with extensive scarring.
May need to lance and drain surgically. 1 1 1 12

13. 1. Staphylococcal Infections (Continued)
Common staphylococcal diseases
E. Impetigo: Problem in hospital nurseries and day care centers. Thin walled vesicles on skin rupture and crust over.
Caused by S. aureus, S. pyogenes, or both.
Highly contagious, spread through direct contact and fomites.
Occurs almost exclusively in children.
Rarely produces fever and easily treated with penicillin. 1 1 1 13

14. Staphylococcal diseases (continued)
6. Scalded skin syndrome: Caused by toxemia from S. aureus strains with two different exfoliative toxins.
Large sheets of bright red skin peel off.
Usually observed in children under 2, but may occur in adults.
Bacteremia and septicemia may occur, and can lead to death within 36 hours.
Require vigorous antibiotic treatment.
Exfoliative toxins are highly antigenic, preventing recurrence. 1 1 1 14

15. Staphylococcal diseases (continued)
7. Toxic shock syndrome (TSS): Fever, vomiting, and sunburnlike rash, followed by shock. Rash later peels.
Presently about 25 cases per year reported.
Most cases are associated with use of superabsorbent tampons.
Males with boils or other staphylococcal infections are at risk.
A few cases associated with use of contraceptive sponge.
5-15% of women have S. aureus in vaginal microflora.
Only a small percentage of these strains produce TSS. 1 1 1 15

16. In addition to skin infections, the staph bacteria can cause:
Bacteremia – a blood infection
Deep abscesses –an abscess that occurs below the skin surface
Endocarditis – an infection on the valves of the heart
Food poisoning – vomiting or diarrhea caused by a staph toxin
Lymphadenitis – an infection of a lymph gland, which causes it to be red, swollen and painful

17. Lymphangitis – an infection of the lymph channels that drain to lymph glands, causing red streaks in the skin
Osteomyelitis – a bone infection
Paronychia – an infection of the skin folds of the nails
Scalded skin syndrome
Septic arthritis – an infection of a joint, like a hip or a knee
Styes – an infection of the glands on the eyelid
Toxic shock syndrome

18. MRSA MRSA stands for Methicillin-Resistant Staphylococcus aureus
MRSA are Staph aureus bacteria that have become resistant to this antibiotic.
Found in the nose, and sometimes on the skin, but also can grow in wounds and other sites of the body
MRSA can no longer be killed by this antibiotic 18

19. 2. Streptococcal Infections
Gram-positive cocci in chains.
Cause many disease including meningitis, pneumonia, sore throat, otitis media, endocarditis childbirth fever, and dental caries.
Produce multiple toxins and virulence factors.
Stretokinases: Dissolve blood clots.
Proteases: Destroy proteins.
Hyaluronidase: Breaks down connective tissue.
Hemolysins: Lyse red blood cells. Alpha, beta, and gamma hemolysis.
Beta hemolytic streptococci are often associated with human disease.
Streptococcus pyogenes = Group A b-hemolytic streptococci.
Infections are often localized, but can produce great damage when they reach deeper tissue. 1 1 1 19

20. Chapter 21: Diseases of Skin and Eyes 1. Staphylococcal Infections
Gram-positive cocci in irregular clusters
Coagulase negative strains make up to 90% of skin microbiota (S. epidermidis). Only pathogenic when skin is broken or through invasive entry.
Coagulase positive strains tend to be pathogenic. Almost all pathogenic S. aureus strains make coagulase. High correlation between ability to produce coagulase and production of damaging toxins:
Leukocidin: Destroys phagocytic white blood cells.
Exfoliative toxin: Responsible for scalded skin syndrome.
Enterotoxins: Affect gastrointestinal tract.
S. aureus is commonly found in nasal passages. 1 1 1 20

21. 1. Staphylococcal Infections (Continued)
Common staphylococcal diseases
A. Folliculitis: Infection of hair follicles (pimples).
B. Sty: Infected eyelash follicle.
C. Boil (Abscess or Furuncle): More serious infection of hair follicle in which pus is surrounded by inflamed tissue. Usually painful and firm.
D. Carbuncle: Aggregate of several infected follicles (boils). May cause fever, chills, malaise, and death if not treated.
Forms a round, hard, deep area of inflammation, typically on neck and back.
Damages surrounding tissue, with extensive scarring.
May need to lance and drain surgically. 1 1 1 21

22. 1. Staphylococcal Infections (Continued)
Common staphylococcal diseases
E. Impetigo: Problem in hospital nurseries and day care centers. Thin walled vesicles on skin rupture and crust over.
Caused by S. aureus, S. pyogenes, or both.
Highly contagious, spread through direct contact and fomites.
Occurs almost exclusively in children.
Rarely produces fever and easily treated with penicillin. 1 1 1 22

23. Staphylococcal diseases (continued)
6. Scalded skin syndrome: Caused by toxemia from S. aureus strains with two different exfoliative toxins.
Large sheets of bright red skin peel off.
Usually observed in children under 2, but may occur in adults.
Bacteremia and septicemia may occur, and can lead to death within 36 hours.
Require vigorous antibiotic treatment.
Exfoliative toxins are highly antigenic, preventing recurrence. 1 1 1 23

24. Staphylococcal diseases (continued)
7. Toxic shock syndrome (TSS): Fever, vomiting, and sunburnlike rash, followed by shock. Rash later peels.
Presently about 25 cases per year reported.
Most cases are associated with use of superabsorbent tampons.
Males with boils or other staphylococcal infections are at risk.
A few cases associated with use of contraceptive sponge.
5-15% of women have S. aureus in vaginal microflora.
Only a small percentage of these strains produce TSS. 1 1 1 24

25. In addition to skin infections, the staph bacteria can cause:
Bacteremia – a blood infection
Deep abscesses –an abscess that occurs below the skin surface
Endocarditis – an infection on the valves of the heart
Food poisoning – vomiting or diarrhea caused by a staph toxin
Lymphadenitis – an infection of a lymph gland, which causes it to be red, swollen and painful

26. MRSA MRSA stands for Methicillin-Resistant Staphylococcus aureus
MRSA are Staph aureus bacteria that have become resistant to this antibiotic.
Found in the nose, and sometimes on the skin, but also can grow in wounds and other sites of the body
MRSA can no longer be killed by this antibiotic 26

27. 2. Streptococcal Infections
Gram-positive cocci in chains.
Cause many disease including meningitis, pneumonia, sore throat, otitis media, endocarditis childbirth fever, and dental caries.
Produce multiple toxins and virulence factors.
Stretokinases: Dissolve blood clots.
Proteases: Destroy proteins.
Hyaluronidase: Breaks down connective tissue.
Hemolysins: Lyse red blood cells. Alpha, beta, and gamma hemolysis.
Beta hemolytic streptococci are often associated with human disease.
Streptococcus pyogenes = Group A b-hemolytic streptococci.
Infections are often localized, but can produce great damage when they reach deeper tissue. 1 1 1 27

28. Common streptococcal skin diseases
Erysipelas: From Greek erythos = red, and pella = skin. Also called St. Anthony’s fire. Common skin infection associated with S. pyogenes.
Spread through contact or insect bites.
Skin erupts into reddish patches with raised margins.
High fever is common.
Organisms can spread through lymphatics and cause septicemia, abscesses, pneumonia, endocarditis, arthritis, and even death if untreated.
Mortality was high before use of antibiotics.
Responds well to antibiotic (b-lactams) treatment. 1 1 1 28

29. Common streptococcal skin diseases
Flesh eating bacterial infections:
Caused by invasive group A streptococci.
15,000 cases per year in U.S.
Exotoxin A acts as superantigen causing damage by the immune system.
Attacks and destroys muscle (myositis), muscle covering (fasciitis), and solid tissue (cellulitis).
Can destroy several inches of tissue per hour.
Antibiotics are not effective because dead tissue has no circulation.
Requires amputation or surgical removal of tissue.
Mortality rate up to 40%
Impetigo: Refer to previous description. 1 1 1 29

30. Flesh eating bacterial infections
Caused by invasive group A streptococci.
15,000 cases per year in U.S.
Exotoxin A acts as superantigen causing damage by the immune system.
Attacks and destroys muscle (myositis), muscle covering (fasciitis), and solid tissue (cellulitis).
Can destroy several inches of tissue per hour.
Antibiotics are not effective because dead tissue has no circulation.
Requires amputation or surgical removal of tissue.
Mortality rate up to 40% 1 1 30 1

31. Bacillus Classification
All are large Gram-positive bacilli
Are aerobic
Form endospores
Most are found in dust and soil
Bacillus anthracis is the major pathogen in the group
Morphology and Cultural Characteristics (Bacillus anthracis)

32. Bacillus Virulence factors
Grow well on ordinary lab media producing large granular colonies with a coarse texture.
Virulence factors
Capsule­ helps organism to resist phagocytosis but antibodies are not protective.
Exotoxin­ is very complex and is produced only when the bacteria is growing in animal tissues.
Toxin production is mediated by a temperature sensitive plasmid.
The toxin consists of three protein components (maximum toxicity occurs when all three components are present).

33. Bacillus Clinical significance
Anthrax which is the disease caused by B. anthracis is essentially a disease of animals who acquire the organism by ingestion or inhalation of spores.
The spores are extremely resistant to adverse chemicals and physical environments.
They may remain a source of infection in soil for 2-3 years.
Man acquires anthrax usually from contact with animal products; less commonly from working in an agricultural setting with infected animals.

34. Bacillus
Man may acquire the organism through skin abrasions, by inhalation of spores, or by ingestion. The disease that develops depends upon the mode of transmission:
Pulmonary (Woolsorter‘s disease)­ Spores are inhaled and germinate in the lungs where they multiply and spread to cause a fatal septicemia or meningitis.
This is the most serious form of the disease.
 Intestinal anthrax­ results from ingestion of spores.

35. Bacillus Antibiotic susceptibility and treatment
Penicillin or tetracycline
A short-term PA vaccine is available for industrial workers and others at high risk.
Other Bacillus species
Bacillus subtilis, and occasional other species may occasionally cause opportunistic infections.
Bacillus cereus is a major cause of enterotoxin food poisoning;
The toxin is protein in nature and can be destroyed by heating;
Food poisoning occurs after ingestion of pre-formed toxin;
Vomiting occurs 1-5 hours after ingestion 

36. Bacillus
B. cereus is also an opportunistic pathogen that has been cultured from cases of septicemia, endocarditis, meningitis, wound infections, pneumonia, and fulminant eye infections
In addition to the enterotoxin that bacteria may produce, a dermonecrotic and a lethal toxin, hemolysins, lecithinase, proteases, and nucleases may be involved in its pathogenesis
Clindamycin with or without gentamycin may be used for treatment of infections

37. Clostridium
Clostridium form endospores under adverse environmental conditions
Spores are a survival mechanism
Spores are characterized on the basis of position, size and shape
Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal (OST) spores
C. tetani have round terminal (RT) spores

38. Clostridium
Clostridium form endospores under adverse environmental conditions
Spores are a survival mechanism
Spores are characterized on the basis of position, size and shape
Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal (OST) spores
C. tetani have round terminal (RT) spores

39. Clostridium perfringens

40. Micro & Macroscopic C. perfringens
NOTE: Large rectangular gram-positive bacilli
Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin
NOTE: Double zone of hemolysis

41. Clostridial Cellulitis

42. Clostridium tetani

43. Clostridium tetani Gram Stain
NOTE: Round terminal spores give cells a “drumstick” or “tennis racket” appearance.

44. Opisthotonos in Tetanus Patient

45. Mechanism of Action of Tetanus Toxin

46. Clostridium botulinum

47. Mechanism of Action of Botulinum Toxin

48. Rates of Isolation of C. botulinum and Botulinum Toxin

49. Tuberculosis (TB, consumption) M. tuberculosis major human disease
healthy people
problems
association with AIDS
multiple drug-resistance

50. M. avium- M. intracellulare complex (M. avium)
non-AIDS
infection almost never
AIDS
major bacterial opportunist
multiple drug-resistance

51. M. bovis
spread from cattle
infected cattle are culled
positive skin test
rarely seen in US

52. M. leprae leprosy major disease of third world
Leprosy rates in Australia are only about one case per million,
but are higher in Aboriginal Australians and immigrants from infected areas.
Worldwide, leprosy is still endemic mostly in Africa and around India.
There is also some leprosy in parts of the USA

53. Laboratory diagnosis - tuberculosis
skin testing
delayed hypersensitivity
tuberculin
protein purified derivative, PPD
X-ray

54. Laboratory diagnosis M. tuberculosis
acid fast bacteria
sputum

55. Positive skin test -tuberculosis
indicates exposure to organism
does not indicate active disease

56. Laboratory diagnosis M. tuberculosis (culture)
grows very slowly
several weeks
non-pigmented colonies
niacin production
differentiates from other mycobacteria

57. Mycobacterium leprae

58. Leprosy (Hansen's Disease)
M. leprae
causative agent
chronic disease
disfigurement
rarely seen in the U.S.
common in third world
millions of cases
infects the skin
low temperature

59. Leprosy tuberculoid few organisms active cell-mediated immunity
lepromatous
immunosuppression

60. Leprosy lepromin skin testing acid-fast stains skin biopsies
clinical picture

61. Corynebacterium diphtheriae

62. Corynebacterium diphtheriae
Gram positive
strict aerobe
pleomorphic (e.g. club-shaped)

63. Diphtheria infection upper respiratory tract (pharynx) pseudomembrane
chocking
bacteria do not spread systemically .

64. Diphtheria toxin B binds to host cell A inhibits protein synthesis
ADP-ribose moiety (NADH) attaches
rare amino acid, diphthamide
elongation factor 2 inhibited

65. C. diphtheriae should not be confused with:
diphtheroids
other corynebacteria
propionibacteria

66. Corynebacterium spp.

67. Listeria monocytogenes
Listeriosis
(invasive disease & non-invasive enteritis)
The organism – G+ve ovoid to rod-shaped bacterium
Widespread in environment
and a non-invasive gastroenteritis.

68. Listeria monocytogenes
Characteristics
- grows in wide range of temperatures (1 to 45o C)
- survives freezing
- aerobic & anaerobic
conditions

69. Listeria monocytogenes
The illness – invasive form
- incubation – 30 days
- flu’-like symptoms, diarrhoea,
vomiting, meningitis, septicaemia,
spontaneous abortion

70. Listeria monocytogenes
The illness – invasive form, continued
- infective dose – 100 to cells
- pregnant women, newborn babies, the elderly & AIDS patients
- Rx – penicillin, ampicillin +/- gentamicin

71. Listeria monocytogenes
The illness – non-invasive
- incubation – 18 hours
- diarrhoea, fever, muscle pain,
headache, abdominal cramps &
vomiting

72. Listeria monocytogenes
The illness – non-invasive
- infective dose – > 100 thou. cells/gm
- all individuals susceptible
- Rx - penicillin, ampicillin +/-
gentamicin

73. Listeria monocytogenes
Sources
- human – person-to-person rare
- animal – diseased animals shed in faeces,
contamination of red meat; silage
- food – ready-to-eat cooked food with long
shelf-life
- raw foods
- environment – widespread in soil, water & sewage
(Hospitals & occupational exposure)

74. Listeria monocytogenes