reading

APPENDIX A PREPARATION OF BLOOD CULTURE BOTTLES

Scope and objective

This document describes the procedure for preparation and quality control (QC) testing of blood culture bottles (adult and pediatric) for medical use.

Equipment

Equipment listed below is necessary to perform this job:

Water still and container for distilled water
Graduated cylinder
Magnetic stirrer
Magnetic hot plate
Electronic balance
Autoclave
Peristaltic pump
Blood bottle vials 50 ml (pediatric) and 100 ml (adults)
Blood bottle cap crimper

Procedure

Wash all the vials in clean water before using.
Print and begin to complete “Form UHS-F03, Preparation of Blood Culture Bottles” with all available information before starting production.
Calculate the correct amount of brain heart infusion (BHI) powder with the formula written on the powder box (X grams for 1 liter) and the SPS amount to add (0.25 g/liter of BHI media).
Choose a bottle with a sufficient volume.
Clean and make sure there are no small particles inside the bottle.
>1 μS/cm; otherwise adjust it with HCl 0.1N.
Add half of the total volume of distilled water needed in the bottle with a graduated cylinder.
Weigh the BHI powder and also the correct amount of SPS.
Transfer the powder into the bottle and add the magnetic bar: adjust the magnetic hot plate to approximately 500 rpm and 100°C.
Mix for 30 min.
Add the rest of the water.
Close the bottle and warm on the hot plate until the medium is completely dissolved.
Set up the peristaltic pump to deliver the correct volume of the solution (25 ml or 50 ml), check the volume on the scale. Dispense 25 ml in pediatric bottles and 50 ml in adult bottles.
Seal bottles with rubber stopper and metal cap using the crimper (Fig. 3).
Place all the bottles in a metal basket.
Bring the basket to the autoclave with the trolley.
Autoclave: fix a cycle of 20 min at 121°C and do not forget to add an autoclave control (see the SOP for autoclave use).
When the autoclave cycle is complete, take out the bottles from the autoclave.
Create the lot number as follows: BHI-ddmmyy.
Example: Brain Heart Infusion media made on January 23, 2013, will be assigned a Lot # BHI-230113.
Assign an expiration date of 6 months from the date of production.
Label each batch with a lot number and expiration date.
Store the blood bottle at room temperature 25 to 30°C.

Figure 3 Blood culture bottle metal cap crimping device.

Quality control

Internal Quality Control (IQC)

Sterility control. Depending on the batch size, follow the sampling plan as outlined in Table 1.

Growth promotion testing

Strains used as positive control

Disinfect the bottle septum with 70% ethanol.
Use a fresh overnight subculture of the two strains.
Prepare a 0.5McFarland solution for each strain by using the reference tube for 0.5McFarland.

Table 1 Sampling plan for sterility control of blood culture bottles from Australian Guidelines (17)

Interpretation: For a batch of 520 bottles, the quality control sample would be 20 bottles.

aFirst Sample: Accept the batch if none of the bottles are contaminated. If 3 bottles are contaminated, reject the batch. If 1 or 2 bottles are contaminated, collect another sample of 20 bottles as second sample. WARNING!! Total the number of contaminated bottles between the first and second samples!

bSecond Sample: Accept the batch if the number of contaminated bottles (sample 1 plus sample 2) is equal to or less than 3 bottles. If the cumulative number of bottles is equal to or greater than 4, reject the batch.

Perform a 10-5 dilution.
Inoculate one bottle from each batch with:
- 100 ml of 10-5 dilution of each organism
- 5 ml of sheep blood
Label the bottle with the date, operator name, and organism name.
Incubate both bottles at 35°C for a maximum of 3 days.
When bottle appears turbid, subculture:
- S. pneumoniae ATCC 49619 on BAP
- H. influenzae ATCC 49247 on chocolate agar
Incubate 24 h in a candle jar.
Check for growth.
Record the data on QC form for: Preparation of Blood Culture Bottles.

Independent External Quality Control (EQC)

Performed at Sihanouk Hospital Center of Hope microbiology laboratory using SOP UHS-05, Enumeration of Microorganisms.

Safety and security

BHI powder: use a N95 mask.
ATCC strains: use a biosafety cabinet.

References

Brain Heart Infusion M210-5kg, HiMedia Laboratories datasheet http://www.theasm.org.au/assets/ASM-Society/Guidelines-for-the-Quality-Assurance-of-Medical-Microbiological-culture-media-2nd-edition-July-2012.pdf

Sodium polyanethol sulfonate (S.P.S.) datasheet FD786-25G HiMedia Laboratories.

Microbiology of Food, Animal Feed and Water — Preparation, Production, Storage and Performance Testing of Culture Media. ISO 11133:2014.

Guidelines for the Quality Assurance of Medical Microbiological Culture Media, Australian Society of Microbiology (17).

Batch Life Process for Blood Culture bottles, prepared by Integrated Quality Laboratory Services.

APPENDIX B STANDARD OPERATING PROCEDURE FOR BLOOD CULTURE

Test summary

Blood culture processing assists in the diagnosis of bacteremia and sepsis. Pathogens include Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae, Streptococcus pneumoniae, and Haemophilus influenzae. Salmonella enterica subsp. enterica serovar Typhi, other Salmonella, and Burkholderia pseudomallei are important blood culture isolates seen in Cambodia.

Principle

Blood is a sterile fluid. Inoculation of blood in prepared media bottles and incubation may detect the presence of organisms in the blood. Growth of an organism allows us to perform identification and susceptibility testing.

Specimen Handling and Preparation

Blood samples are inoculated into blood culture media bottles.
It is encouraged to collect two separate venipuncture collections per patient.
Ten milliliters of blood for each adult collection (20 ml total in two bottles) and 2 to 5 ml in one bottle for a pediatric collection).
It is important to use aseptic technique when collecting blood for blood culture so that skin flora or environmental organisms are not introduced into the bottles.
Refer to blood culture collection procedures. DMDP document code: 084 and training material video blood culture collection procedure.

Quality Control

Appropriate QC testing should be performed for all media used, biochemical testing, Gram stain, and antimicrobial susceptibility test (AST) performed according to the laboratory QC procedures.

Reagents, materials, and equipment

Adult and pediatric blood culture bottles
Venting needles
Media for subculture of blood from blood culture bottles:
- Sheep blood agar (BAP)
- Chocolate agar (CHOC)
- MacConkey agar (MAC)
Glass slides for Gram stain preparation
Usual laboratory equipment and consumables

Procedure

Ensure that the information on the request form and bottle match.
Label each blood culture bottle from the same patient, same day and time with the same number, but differentiate each bottle with “A” or “B.”
Record patient details on the worksheet.
Weigh the bottle and record weight on the bottle. Also record uninoculated weight on the worksheet.
Note: All blood culture bottle weights are recorded on the bottle when they are received in the laboratory. Both the uninoculated weight and the inoculated weight should be recorded on the bottle and the worksheet.
Clean the top of the blood culture bottle with 70% alcohol.
Aseptically insert a venting needle.
Incubate bottles for 7 days at 35 to 37°C.
Keep the venting needle in place for the full 7 days of incubation.
Do not disturb the bottle, but check the bottles daily for signs of growth: turbidity, bubbles, hemolysis. If none seen, gently rotate the bottle on the bench to resuspend the blood and mix it. Reincubate until the next day.
Hold for 7 days before reporting final report as “negative.”
Gram stain and subculture all bottles that show signs of growth.
Subculture all bottles after 1 night incubation.
Subculture:
Place 1 drop of blood to CHOC and 1 drop for Gram stain.
- If Gram-positive cocci are detected on the Gram stain, add BAP and an optochin disc to the second quadrant of the BAP plate.
- If Gram-negative bacilli are detected on Gram stain, subculture blood to a BAP and MAC plate. Add Polymyxin B and Gentamycin, Amox/Clav to BAP plate if patient suspected of a melioidosis or thin Gram-negative rod with bipolar staining.
Incubate BAP and CHOC plates in a candle jar. Incubate the MAC plate and the candle jar for 3 days at 35 to 37°C.
Read all agar plates daily for 3 days.
After the visual checks of blood culture bottles, gently invert before reincubation. Examine all bottles daily for turbidity, gas, etc. If any bottles look suspicious, perform a Gram stain and CHOC agar subculture. Hold these subcultures for 3 days and examine daily.
At the end of the 7-day incubation period, perform Gram stain.
Record daily on the specimen worksheet all actions performed.

Reading Gram Stains

Read the Gram stains the same day that they are prepared.
A positive Gram stain result is a critical result. The result may have direct impact on the treatment of the patient. Record all Gram stain results on the specimen worksheet.
Phone the result to the clinician or ward. Record notification on the worksheet, and document in laboratory information system as a preliminary report. Be sure to include the name of the person who took the report, have them read the report back to you, and record the time and date and your initials on both the worksheet and in the computer.

Reading Cultures

Read all plates daily and record results on the specimen worksheet.
If growth is detected, perform identification and susceptibility testing.
Refer to flow charts for pathogen identification.
Refer to the current AST Guidelines to help choose appropriate antibiotic discs for testing.
Keep plates until a final report has been sent to the clinician.
Inoculate a broth for storage of all blood culture isolates at –20°C.

Result Reporting

Blood culture showing “growth”

Primary Report: Gram stain result. Example: Gram-negative bacilli
Secondary Report: add organism identification. Example: E. coli
Final Report: add Antibiotic Susceptibility Test result.
Refer to the current AST Guidelines to help choose appropriate antibiotic discs for reporting.
In some cases, the significance of the isolate will not be clear. In this case, add the following comment: “Significance depends on clinical assessment.”

Blood culture showing “NO growth”

Expected Values: Blood is a sterile fluid.

Note: Growth from a blood culture may indicate bacteremia or sepsis.

Notes

It is important to inoculate the correct volume of blood into a blood culture bottle. Follow the collection procedure. Insufficient blood volume decreases the sensitivity of this test. The uninoculated and inoculated weights will be used to determine the volume of blood that was collected.
Sometimes contaminants from the environment or from skin flora may be introduced to the blood culture bottles. Always report positive growth. Add the comment: “Significance depends on clinical assessment.”
On visual inspection, bottles may appear turbid but show no growth on subculture.
In these circumstances, always carefully examine the Gram stain. If there is any doubt, repeat the Gram stain and subculture.
Some fastidious bacteria may not grow under the usual culture conditions. Some bacteria require a microaerophilic atmosphere (Campylobacter or Helicobacter).
In Cambodia, we do not use anaerobic blood culture bottles or culture for anaerobes.
Sometimes a bottle may not appear turbid, but the subculture will be positive.
Collection of only one blood culture bottle makes the interpretation of the significance of the isolation of skin flora difficult. Communication with clinician should be done before sending the report out.

References

Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. 2007. Manual of Clinical Microbiology. 9th edition. ASM Press, Washington, DC.

References

1.Findlen P. 2004. Introduction: the last man who knew everything…or did he? Antanasius Kircher, S.J. (1602-80) and his world, p 1–48. In Findlen P (ed), Athanasius Kircher. The Last Man Who Knew Everything. Routledge, London, NY.

2.Baldwin M. 2004. Reverie in time of plague. Athanasius Kircher and the plague epidemic of 1656, p 1–48. In Findlen P (ed), Athanasius Kircher. The Last Man Who Knew Everything. Routledge, London, NY.

3.Torrey HB. 1938. Anthanasius Kircher and the progress of medicine. Osiris 5:246–275.

4.Wilson C. 1951. Anamalcula and the theory of animate contagion, p 140–175. In Wilson C (ed), The Invisible World: Early Modern Philosophy and the Invention of the Microscope. Princeton University Press, Princeton NJ. [Third printing, 1997.]

5.Wilson C. 1951. Instruments and applications, p 70–102. In Wilson C (ed), The Invisible World: Early Modern Philosophy and the Invention of the Microscope. Princeton University Press, Princeton NJ. [Third printing, 1997.]

6.Frischknecht F, Renaud O, Shorte SL. 2006. Imaging today’s infectious animalcules. Curr Opin Microbiol 9:297–306.

7.Villemin JA. 2015. On the virulence and specificity of tuberculosis. Int J Tuberc Lung Dis 19:256–266.

8.Hiss PH, Zinsser H. 1911. The bacteriological examination of materials from patients, p 178–180. In Hiss PH, Zinsser H (ed), A Text-Book of Bacteriology: A Practice Treatise for Students and Practitioners of Medicine. D Appleton and Co, New York, NY.

9.Hiss PH, Zinsser H. 1911. Bacilli of the colon-typhoid-dysentery group: the bacillus of typhoid fever, p 399–427. In Hiss PH, Zinsser H (ed), A Text-Book of Bacteriology: A Practice Treatise for Students and Practitioners of Medicine. D Appleton and Co, New York, NY.

10.Muir R, Ritchie J. 1927. Methods of examining the properties of serum – preparation of vaccines – inoculation of animals – methods of obtaining pathological materials for examination, p 122–149. In Muir R, Ritchie J (ed), Manual of Bacteriology, 8th ed. Humphrey Milford Oxford University Press, London, UK.

11.Clough MC. 1917. The cultivation of tubercle bacilli from the circulating blood in military tuberculosis. Am Rev Tuberc 1:598–621.

12.Stuart RD. 1948. The value of Liquoid for blood culture. J Clin Pathol 1:311–314.

13.Von Haebler T, Miles AA. 1938. The action of sodium polyanethol sulphonate (“liquoid”) on blood cultures. J Pathol Bacteriol 48:245–252.

14.Stitt ER, Clough PW, Clough MC. 1938. Blood cultures, p 702–705. In Stitt ER, Clough PW, Clough MC (ed), 9th edition of Practical Bacteriology, Haematology and Parasitology. P. Blakiston’s Son and Co, Inc, Philadelphia, PA.

15.McBurney R. 1952. Bacteriological examination of blood and feces, p 559–592. In Miller SE (ed), A Textbook of Clinical Pathology. The Williams & Wilkins Co, Baltimore, MD.

16.Scott EG. 1951. A practical blood culture procedure. Am J Clin Pathol 21:290–294.

17.Castaneda MR. 1947. A practical method for routine blood cultures in brucellosis. Proc Soc Exp Biol Med 64:114–115.

18.Levin GV, Heim AH, Clendenning JR, Thompson MF. 1962. “Gulliver”--a quest for life on Mars: radioisotopes are used in a miniature instrument designed to detect life during early probes of the planet. Science 138:114–121.

19.Winn WR, White ML, Carter WT, Miller AB, Finegold SM. 1966. Rapid diagnosis of bacteremia with quantitative differential-membrane filtration culture. JAMA 197:539–548.

20.Finegold SM, White ML, Ziment I, Winn WR. 1969. Rapid diagnosis of bacteremia. Appl Microbiol 18:458–463.

21.DeLand FH, Wagner HN Jr. 1969. Early detection of bacterial growth, with carbon-14-labeled glucose. Radiology 92:154–155.

22.DeBlanc HJ Jr, DeLand F, Wagner HN Jr. 1971. Automated radiometric detection of bacteria in 2,967 blood cultures. Appl Microbiol 22:846–849.

23.Gröschel D, Hopfer RL, French JE. 1979. Blood cultures with the BACTEC 225 radiometric microbial growth detection system. Zentralbl Bakteriol [Orig A] 244:316–323.

24.Phillips LE, Martin RR, Gentry LO, Rogers TE. 1979. Assay of serum tobramycin levels with the Bactec 460. Antimicrob Agents Chemother 16:463–467.

25.Courcol RJ, Fruchart A, Roussel-Delvallez M, Martin GR. 1986. Routine evaluation of the nonradiometric BACTEC NR 660 system. J Clin Microbiol 24:26–29.

26.Schnur ER, Azimi PH, Belchis DA. 1989. Poor performance of BACTEC NR 730 blood culture system in early detection of Neisseria meningitidis. J Clin Microbiol 27:654–656.

27.Nolte FS, Williams JM, Jerris RC, Morello JA, Leitch CD, Matushek S, Schwabe LD, Dorigan F, Kocka FE. 1993. Multicenter clinical evaluation of a continuous monitoring blood culture system using fluorescent-sensor technology (BACTEC 9240). J Clin Microbiol 31:552–557.

28.Arpi M, Bremmelgaard A. 1993. Comparison of detection speed and yield in BACTEC NR-860 and Roche BCB blood culture systems. APMIS 101:545–550.

29.Riedel S, Eisinger SW, Dam L, Stamper PD, Carroll KC. 2011. Comparison of BD Bactec Plus Aerobic/F medium to VersaTREK Redox 1 blood culture medium for detection of Candida spp. in seeded blood culture specimens containing therapeutic levels of antifungal agents. J Clin Microbiol 49:1524–1529.

30.Dorn GL, Haynes JR, Burson GG. 1976. Blood culture technique based on centrifugation: developmental phase. J Clin Microbiol 3:251–257.

31.Dorn GL, Burson GG, Haynes JR. 1976. Blood culture technique based on centrifugation: clinical evaluation. J Clin Microbiol 3:258–263.

32.Wallis C, Melnick JL, Wende RD, Riely PE. 1980. Rapid isolation of bacteria from septicemic patients by use of an antimicrobial agent removal device. J Clin Microbiol 11:462–464.

33.Thorpe TC, Wilson ML, Turner JE, DiGuiseppi JL, Willert M, Mirrett S, Reller LB. 1990. BacT/Alert: an automated colorimetric microbial detection system. J Clin Microbiol 28:1608–1612.

34.Morello JA, Leitch C, Nitz S, Dyke JW, Andruszewski M, Maier G, Landau W, Beard MA. 1994. Detection of bacteremia by Difco ESP blood culture system. J Clin Microbiol 32:811–818.

35.Thomson RB Jr, File TM Jr, Tan JS, Evans BL. 1987. Yield, clinical significance, and cost of a combination BACTEC plus Septi-Chek blood culture system. J Clin Microbiol 25:819–823.

36.Trombley C, Anderson JD. 1987. SIGNAL blood culture system for detection of bacteremia in neonates. J Clin Microbiol 25:2098–2101.

37.Stevens M, Patel H, Walters A, Burch K, Jay A, Dowling N, Mitchell CJ, Swann RA, Willis AT, Shanson DC. 1992. Comparison of Sentinel and Bactec blood culture systems. J Clin Pathol 45:815–818.

38.Aubert G, Vautrin AC, Michel VP, Fresard A, Dorche G. 1993. Evaluation of three automated blood culture systems. Bio Argod, Bact T/Alert, bactec NR-860. Pathol Biol (Paris) 41:434–440. (In French.)

39.Stevens CM, Swaine D, Butler C, Carr AH, Weightman A, Catchpole CR, Healing DE, Elliott TS. 1994. Development of o.a.s.i.s., a new automated blood culture system in which detection is based on measurement of bottle headspace pressure changes. J Clin Microbiol 32:1750–1756.

40.Wilson ML, Mirrett S, McDonald LC, Weinstein MP, Fune J, Reller LB. 1999. Controlled clinical comparison of bioMérieux VITAL and BACTEC NR-660 blood culture systems for detection of bacteremia and fungemia in adults. J Clin Microbiol 37:1709–1713.

References

1.Baron EJ, Weinstein MP, Dunne WM Jr, Yagupsky P, Welch DF, Wilson DM. 2005. Cumitech 1C, Blood Cultures IV. Baron EJ, Coordinating ed. ASM Press, Washington, DC.

2.Wilson ML, Mitchell M, Morris AJ, Murray PR, Reimer LG, Reller LB, Towns M, Weinstein MP, Wellstood SA, Dunne WM Jr, Jerris RC, Welch DF. 2007. Principles and Procedures for Blood Cultures; Approved Guideline M47-A. CLSI, Wayne, PA.

3.Riedel S, Bourbeau P, Swartz B, Brecher S, Carroll KC, Stamper PD, Dunne WM, McCardle T, Walk N, Fiebelkorn K, Sewell D, Richter SS, Beekmann S, Doern GV. 2008. Timing of specimen collection for blood cultures from febrile patients with bacteremia. J Clin Microbiol 46:1381–1385.

4.Dunne WM Jr, LaRocco M. 2001. Blood culture systems, p 189–209. In Cimolai N (ed), Laboratory Diagnosis of Bacterial Infections. Marcel-Dekker, New York, NY.

5.Schaffner J, Chochua S, Kourbatova EV, Barragan M, Wang YF, Blumberg HM, del Rio C, Walker HK, Leonard MK. 2009. High mortality among patients with positive blood cultures at a children’s hospital in Tbilisi, Georgia. J Infect Dev Ctries 3:267–272.

6.Campbell J, Washington JA II. 1980. Evaluation of the necessity for routine terminal subcultures of previously negative blood cultures. J Clin Microbiol 12:576–578.

7.Gill VJ. 1981. Lack of clinical relevance in routine terminal subculturing of blood cultures. J Clin Microbiol 14:116–118.

8.Blazevic DJ, Stemper JE, Matsen JM. 1974. Comparison of macroscopic examination, routine gram stains, and routine subcultures in the initial detection of positive blood cultures. Appl Microbiol 27:537–539.

9.Doern GV, Brueggemann AB, Dunne WM, Jenkins SG, Halstead DC, McLaughlin JC. 1997. Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system. J Clin Microbiol 35:1290–1292.

10.Han XY, Truant AL. 1999. The detection of positive blood cultures by the AccuMed ESP-384 system: the clinical significance of three-day testing. Diagn Microbiol Infect Dis 33:1–6.

11.Castaneda MR. 1947. A practical method for routine blood cultures in brucellosis. Proc Soc Exp Biol Med 64:114–115.

12.Murray PR, Niles AC, Heeren RL, Curren MM, James LE, Hoppe-Bauer JE. 1988. Comparative evaluation of the oxoid signal and Roche Septi-Chek blood culture systems. J Clin Microbiol 26:2526–2530.

13.Daley C, Lim I, Modra J, Wilkinson I. 1990. Comparative evaluation of nonradiometric BACTEC and improved oxoid signal blood culture systems in a clinical laboratory. J Clin Microbiol 28:1586–1590.

14.Rohner P, Pepey B, Auckenthaler R. 1995. Comparison of BacT/Alert with Signal blood culture system. J Clin Microbiol 33:313–317.

15.Weinstein MP, Mirrett S, Wilson ML, Harrell LJ, Stratton CW, Reller LB. 1991. Controlled evaluation of BACTEC Plus 26 and Roche Septi-Chek aerobic blood culture bottles. J Clin Microbiol 29:879–882.

16.Welby PL, Zusag TM, Storch GA. 1992. Comparison of the Bactec Peds Plus pediatric blood culture vial with Roche pediatric Septi-Chek for blood cultures from pediatric patients. J Clin Microbiol 30:1361–1362.

17.Culture Media Special Interest Group for the Australian Society for Microbiology. 2012. Guidelines for Assuring Quality of Medical Microbiological Culture Media. The Australian Society for Microbiology, Melbourne, Victoria, Australia.