Lab 9 Wound Specimens

Question 1

What are the main three layers of the skin? Describe each.

Answer 1

Epidermis- a superficial layer, consisting of a horny layer, a granular layer and a prickle cell layer

Dermis- separated from the epidermis by a basal cell layer; containing specialized structures and functions – e.g. hair shafts, nerve endings, sebaceous glands, veins, arteries, erector pili, muscles.
- the hair follicles, sebaceous glands and sweat glands open to the skin surface through the epidermis

Subcutaneous fat- a layer of fat which sits between the skin and the thin fascial membranes which cover muscles, ligaments and other connective tissues.

Question 2

Why is the skin prone to infection?

Answer 2

Because the skin is the most accessible organ of the body, it is the one most readily traumatized and therefore the one subjected most frequently to the risk of infection.

Question 3

What layer is under the subcutaneous fat layer and what is its importance?

Answer 3

The importance of the fascia is that it is a barrier that determines the extent to which infections may spread but may also create challenges to therapy, due to its impermeability, which may have to be met surgically ie. Debridement rather that ‘drugs’.

See Fig. 75.1 of textbook.

From web:
Debridement: A procedure to remove debris or infected/dead tissue from a wound.

Question 4

What factors affect what normal flora is on the skin?

Answer 4

Factors that control the skins’ microbial load include the following:

1. The limited amount of moisture present
2. Acid pH of normal skin
3. Surface temperature (less than optimum for many pathogens)
4. Salty sweat
5. Excreted chemicals ( sebum, fatty acids, urea)
6. Competition between different species of the normal flora

An alteration in any of these factors upsets the balance of the commensal flora, and predisposes to infection.

Question 5

What are the two types of skin infections?

Answer 5

Infections may be classified as primary and secondary.

Primary- arising from organisms or extracellular products (eg. toxins) that reach the skin through the blood as part of systemic disease (no obvious portal of entry eg. erysipelas- an acute, cutaneous inflammatory disease caused by ß-hemolytic streptococci)

Secondary- arising from complications of injury to the skin such as penetrating wounds, surgical trauma, or abrasions. These can be either monomicrobial, as in staphylococcal wound infection, or poly-microbial, as in some gangrenous conditions caused by microaerophilic streptococci and anaerobes. These infections may also be localized or extensive, depending on the extent of the underlying disease or precipitating trauma.

Question 6

What are examples of superficial erythematous infections?

Answer 6

Ulcers, nodules, sinus tracts, burns, simple post operative wounds, wound infections, and bites.

Refer Table 3.13 in lab notes.

Question 7

What are unacceptable specimens for wounds?

Answer 7

Unacceptable specimens
1. Mislabeled etc.; lack of specimen date or specimen information
2. Dry swab
3. Specimen frozen
4. Too old (too long in transport): >24 hours
5. Specimen leaking
6. Specimen in formalin

Question 8

What is the ideal collection method for samples from wounds? What is often done instead?

Answer 8

Superficial wounds should be ideally collected by aspiration, if there is enough pus in a wound area.

If aspiration is not possible, often swabs are collected.

Question 8

When is only aerobic testing done for wounds? When is anaerobic testing appropriate?

Answer 8

If the specimen is considered superficial, only aerobic culture is appropriate, but if it is an aspiration of a deeper wound or an abscess or a bite wound, anaerobic culture might be requested and should be performed.

Question 8

What transport media is used for wound specimen swabs?

Answer 8

Amies or Stuart’s transport media.

Question 9

What are the most commonly isolated organisms on superficial wounds? (not animal bites)

Answer 9

Staph. aureus (commonly MRSA) and beta hemolytic Streptococcus (A, B, C, G).

Question 10

If a wound is caused by an animal bite what organisms might be suspected?

Answer 10

Animal bites –> might find Pasteurella multocida, Capnocytophaga spp.,
Eikenella corrodens and anaerobes (if they are deep specimens).

Question 11

If abscesses or fluids have access to the mucosas or GI tract due to an injury what organisms may be involved in the cause of infection?

Answer 11

Any organism in the mucosas or GI tract can be the cause of infection, frequently a mix of bacteria (aerobic and anaerobic).

Question 12

What plates are planted typically for wound specimens?

Answer 12

Plant into
1. BA and Mac (routine)
2. CA added for bites, abscesses and genital sites.

If swab, label a tube with 0.5 mL broth (TSB) or saline. Elute swab in 0.5 mL TSB/saline (for even amount of sample in plates).

Question 13

What is the method for creating a direct smear?

Answer 13

1. Use pencil on the frosted end of an alcohol-cleaned slide
2. Draw a large circle at the back with a grease pencil for ease of focusing.
3. Add one drop of eluted specimen over the encircled area of the slide, air dry and Gram stain.

Question 14

How are the plates inoculated?

Answer 14

1. Plant one drop of specimen from eluted broth into each plate,
2. Do not invert the plates until drop has been distributed onto first quadrant to prevent the ‘wet’ inoculum from running over the plate.
3. Streak for isolation.
4. Incubate Choc in CO2, BA and Mac at 35ºC aerobically.

Question 15

How is the gram-stained smear examined for wound specimens?

Answer 15

1. Evaluate relative numbers of PMN’s , indicative of infection
2. Evaluate relative quantities of different microorganisms (1+ to 4+).
3. Report as direct Gram smear on requisition/worksheet.

Question 16

How do examine and work up the plates from wound specimens?

Answer 16

Examine for growth at 24 and 48h (up to 72 hours if NG or CA was planted, i.e. specimens from genital area and incubated at least for 72 hours in CO2).

1. Examine all media for growth
2. If no growth is present, report as: No growth at 24 hours, FRTF
3. If growth is observed:
   a) record quantity (1+ to 4+) and describe the characteristics of each colony type seen
   b) perform Gram stains as needed
   c) perform basic screen or ‘spot’ tests to rule out potential contaminants and/or suspect the presence of a potential pathogen
   d) perform any identification tests on well-isolated colonies of potential pathogens or API or sub if necessary.
   e) perform susceptibility tests as necessary (if possible)
   f) re-incubate plates from which colonies have been worked up.
   g) Report “preliminary report” if applicable

Question 17

What organisms are always worked up in any amount?

Answer 17

Probable pathogens such as
1. Staph. aureus
2. Beta hemolytic Streptococcus, 3. Pseudomonas aeruginosa,
4. Pasteurella multocida,
5. Eikenella spp,
6. Capnocytophaga spp,
7. Vibrio spp,
8. Aeromonas spp

interesting fact: all GNB on this list are oxidase POSITIVE, so an oxidase could be performed to rule them in/out

Question 18

What organisms are only worked up if predominant with one other organism present (two in total) and in 3-4+ amount?

Answer 18

Potential pathogens: other GNB, Enterococcus
- with unpredictable antimicrobial susceptibility/ resistance patterns

Question 19

How are probable skin contaminants worked up or not? Which ones are they? When do you work them up?

Answer 19

List (meaning just mention) with NO susceptibility results:
1. Coagulase-negative staph,
2. Corynebacterium spp.,
3. viridans streptococci,
4. Bacillus spp.,

**Unless they are pure and in high amounts (3-4+).

Question 20

What are the post-analytical considerations for wound specimens, i.e. reporting, etc.?

Answer 20

1. Interpret and record the results of subcultures and identification / susceptibility tests
2. Perform any additional testing if appropriate
3. Create a report based on the previous protocol (I think she means guidelines on how much to work up the organisms).
4. Critical reporting for these specimens: ONLY if related to a reportable antimicrobial susceptibility (MRSA-VRE-ESBL, etc.)

Question 21

Where is Pasteurella spp. found typically in animals and humans?

Answer 21

1. Nasopharynx and GI tract of domestic and wild animals.
2. Upper respiratory tract of animal handlers due to them breathing in the bacteria excrement.
3. In humans, it is associated with focal infections following animal bites, chronic pulmonary disease, and systemic diseases including meningitis.

Whenever there is an animal association in the clinical history, this pathogen should be considered as one of the suspects.

Question 22

Does a susceptibility testing need to be done on infections caused by P. multocida?

Answer 22

P. multocida are typically susceptible to Penicillin, which marks a difference with similar genera that are always intrinsically resistant. Therefore, antimicrobial testing specially for superficial infections is not required.

Question 23

What is the first clue to identify P. multocida (as they are no specific procedures to ID them)?

Answer 23

Morphology in the Gram stained smear (very short Gran negative coccobacilli - see below), along with the specimen source and association with animals.

Gram Stain: Short straight bacilli (with bipolar staining = “Safety pins”), too short sometimes (tricky…might confuse with cocci). May have a capsule.

Question 24

Describe the colonial morphology of P. multocida on BA, CA, and MA

Answer 24

BA: Mucoid or wet gray opaque colony with “mousy” smell

CA: might grow a lot “healthier” due to the CO2 incubation.

MAC: No growth!!

Question 25

What are some chemical tests that can be performed and their results for P. multocida?

Answer 25

Catalase: Positive
Oxidase: Weak positive (use generous inoculum= might give false NEG’s)
Glucose utilization: TSI positive (A/A/-), but it takes time…

Question 26

When is it justified to perform API NH on a suspected P. multocida colony?

Answer 26

API NE (and other automated systems) will help to ID as long as it is suspected and oxidase is positive.

Question 27

What organism can cause flesh eating disease?

Answer 27

Strep. pyogenes

Small pinprick or scratch allows bacteria to get under skin. Pain disproportionate to the injury (feels like a pulled muscle) usually occurs within 24 hours. Flu-like symptoms follow and purplish blisters begin to form. Without immediate antibiotics and surgical removal of infected tissue, toxic shock sets in and then it can be potentially fatal.