1. General Medical Microbiology
Introduction

2. Specimen Collection
The failure to isolate the causative agent of an infectious disease is frequently the result of faulty collecting or transport techniques. Therefore, when collecting a specimen for microbiological examination, the primary general considerations that need to be addressed are:

3. General Considerations
The specimen obtained must be representative of the disease process. For example, a throat swab can’t substitute for sputum in cases of pneumonia. WHY?
Sufficient material must be collected to ensure a complete and accurate exam.
The specimen must be obtained in a manner that avoids contamination by the patient’s own normal flora

4. General Considerations
If at all possible, the specimen should be obtained before initiating antibiotic therapy.
It is best to obtain the specimen at the acute phase of the disease when the causative organism is most likely to be found in large numbers.
The specimen should be delivered promptly to the laboratory. WHY?

5. General Considerations
The laboratory should be provided with sufficient clinical information to guide the microbiologist in the selection of the suitable media and incubation conditions. (Some organisms require special media for growth, while other organisms require special incubation conditions).
The following specimens should normally be sterile (i.e., have no normal flora):

6. General Considerations
Blood
Cerebral spinal fluid (CSF)
Tissue
Serous fluids
Specimens from the lower respiratory tract
Urine taken directly from the bladder or kidney
The following specimens are likely to be contaminated with normal flora:
Specimens from the upper respiratory tract, including the mouth and nose

7. General Considerations
Sputum
Feces
Genital tract specimens
Skin
The following specimens may be contaminated with normal flora, but the numbers of contaminating organisms are likely to be quite low:
Conjunctiva of the eyes
External ears

8. Circumventing Normal Flora
There are several different ways that one may use to circumvent problems of contamination with indigenous normal flora:
Antisepsis – antiseptics such as tincture of iodine should be applied to the skin prior to aspiration of abscesses and various normally sterile body fluids such as blood and CSF

9. Circumventing Normal Flora
Decontamination – procedures that selectively inhibit or destroy microorganisms other than those of specific interest may be used. For example sputum may be treated with NaOH prior to culturing for mycobacteria
Use of selective media – use media that selectively inhibits growth of normal flora and allows for growth of pathogens, i.e. MSA (more on this later)

10. Circumventing Normal Flora
Quantification – the classic example of quantification procedures is the procedure used for urine specimens. A calibrated loop that delivers 1 ul of urine (1/1000 of a ml) is used and if the colony count is > 100 colonies, the result is considered to be significant and indicative of infection (How many organisms would this be/ml?)

11. Urine colony counts

12. Circumventing Normal Flora
Microscopy –
A cytological exam should be done to look for the presence of squamous epithelial cells in urine, sputum, or wound specimens which, when present, indicate likely contamination with skin or mucosal flora.
A new specimen should be requested when numerous squamous epithelial cells are present in the specimen.

13. Inappropriate sputum specimen

14. Good sputum specimen

15. Circumventing Normal Flora
Invasive procedures that allow the physician to avoid normal flora in collecting the specimen including:
Transtrachael aspirate
Suprapubic aspirate
Bronchoscopy
Needle biopsies

16. Transtrachael aspirate

17. Suprapubic aspirate

18. General instructions for collection of specimens
General instructions for specimen collection:
Identification – the specimen should be labeled with the following:
Patient’s name and identification number
Patient’s location
Patient’s physician
Site/source of specimen
Type of exam requested (bacterial, fungal, viral, parasites)
Tentative diagnosis (Why is this important?)
Date and time of specimen collection (Why is this important?)
If antibiotics have been administered the type, dosage and time given should be provided

19. General instructions for collection of specimens
Transport containers, devices, media:
Swabs are convenient and economical, but:
They are often inadequate in terms of the amount of specimen required for multiple types of culturing.
The recovery of bacteria is usually< 10% of the original inoculum.
Swabs are often used for throat cultures and for cervical, vaginal and urethral secretions.
They should not be used when pus or exudate is available, for surgical specimens, or for cultures for anaerobes or mycobacteria.

20. General instructions for collection of specimens
Syringes – these are good for aspirates. The tip of the needles should be plugged with a sterile stopper or the needle should be removed and the syringe capped.
Tubes, bottles, and jars should be sterile and leak proof.
Fecal transport systems – polyvinyl alcohol fixative should be used for preservation of fecal parasites
For sexually transmitted diseases it is best to inoculate media directly at the bedside of the patient or to use a swab/transport media system specifically designed for optimal recovery of sexually transmitted pathogens.
For specimens for viral examination or for anaerobic culturing, appropriate transport media should be used.

21. General instructions for collection of specimens
The specimen must be promptly transported to the laboratory to preserve the viability of fastidious organisms and to prevent overgrowth of fastidious organisms by more rapidly growing bacteria which may be insignificant.
Sometimes refrigeration of the specimen is warranted (i.e. urine specimens),
Sometimes refrigeration will kill fastidious organisms (i. e. Streptococcus pneumoniae or Neisseria gonorrhoeae from sputum or genital tract specimens, respectively)

22. Specific guidelines Specific guidelines for specimen collection:
Septicemia means that organisms, or their toxins are present, and growing in the blood.
Bacteremia simply means the presence of organisms in the blood without causing infection (Can you think of examples where you might find this?)
For septicemia, 2-3 cultures should be collected by venipuncture in a 24 hour period
20-30 mls should be collected for each sample.
The samples should be inoculated into culture media directly at the bedside of the patient.

23. Venipuncture

24. Specific guidelines Infections of other normally sterile body fluids
Meningitis and encephalitis – collect CSF via a lumber puncture
Pleural, pericardial and synovial fluids – aspirate and collect sufficient quantities for a thorough exam.
Wounds
The best specimens are aspirates of pus or exudate.
A swab is usually not a good way to collect specimens from wounds.

25. Lumbar puncture

26. Specific guidelines
Upper respiratory tract infections – a swab is sufficient for collecting URT specimens
Lower respiratory tract infections – collect sputum. Alternatively, may use:
Transtrachael aspiration,
Bronchial wash, or
Lung puncture for collecting the specimen.
Urinary tract infection
Use a clean voided midstream specimen

27. Clean voided midstream

28. Specific guidelines
Use catherization
Use suprapubic aspiration of bladder or kidney
Gastroenteritis – collect a stool sample in a sterile container.
For intestinal parasites, three separate specimens should be collected because some organisms are only present intermittently.
Genital tract infections – a swab or aspirate of exudate plus direct inoculation on media is best
Ocular infections – a swab is sufficient
Tissue specimens – that should be obvious!

29. Direct exam Direct examination of specimens
Gross examination of the specimen may yield important information:
If CSF is cloudy this probably indicates infection
Sputum – the color, consistency and odor may give clues as to the causative agent of infection
If the stool contains mucous and blood, this is typical of dysentery
If the odor is foul, the specimen may contain anaerobes
Visible granules (which are aggregates of actinomycetes) are found in actinomycete infections

30. Initial microscopic examination
Differential stains
Gram stain –
Allows one to determine if organisms are Gram positive or Gram negative (Why would this be important for a physician?)
To determine the shape of bacteria it is usually necessary to use oil. On low power it is possible to see fungi, some parasites and WBCs. (The hallmark of an acute bacterial infection is numerous PMNs).
Positive and negative controls should always be done (Why?)
A Gram stain of a direct smear can provide important information for some specimens, but is useless for others (Give examples of each)
It is important to not over interpret Gram stain results

31. Gram stain of Bacillus species

32. Gram stain of Staphylococcus aureus

33. Gram stain of Neisseria species

34. Gram stain of Haemophilus species

35. Initial microscopic examination
Acid fast stain –
Divides organisms into acid-fast and non acid-fast organisms
Acid fast staining results are clinically very important for diagnosing tuberculosis. Mycobacteria tuberculosis grows so slowly that it may be 6-8 weeks before a culture report is signed out. It is important that the physician know if acid-fast bacilli are seen in the direct smear so that appropriate antimicrobic therapy can be initiated as soon as possible.

36. Acid fast stain

37. Initial microscopic examination
Special stains
Spore stain The position of the spore may be diagnostically important

38. Initial microscopic examination
Capsule stain – usually the background and the organism are stained while the capsule is left unstained.

39. Initial microscopic examination
Trichrome stain – for permanent stained smears of intestinal parasites:

40. Initial microscopic examination
Iron-hematoxylin stain – another way to make make permanent stained smears of intestinal parasites

41. Initial microscopic examination
Wrights and Giemsa stains of blood – parasites and bacteria in the blood may be seen

42. Initial microscopic examination
Wet mounts
India ink mount for Cryptococcus neoformans

43. Initial microscopic examination
Lactophenol cotton blue – to observe fungi

44. Initial microscopic examination
10% KOH – to observe fungi from skin scrapings. The KOH destroys the epithelial cells without harming the fungal elements.

45. Initial microscopic examination
Iodine stain – used for stool examination for parasites