Friday, July 13, 2007


Topic: Blood Culture

Posted by: Azhar Hamdan TG01 (0503269C)

Equipments & Materials:

  • BACTEC™ Fluorescent series blood culture system
  • BACTEC™ Plus Aerobic/F Culture Vials Soybean-Casein Digest Broth (Blue Cap)
  • BACTEC™ Plus Anaerobic/F Culture Vials Soybean-Casein Digest Broth (Gold Cap)
  • Aerobic venting unit
  • Class II Biosafety Cabinet
  • Calibrated inoculating loops
  • Blood Agar Plate (BAP)
  • MacConkey Agar (MAC)
  • CDC Anaerobic Blood Agar (CDC)
  • Sterile 70% ethanol pads (i.e. Alcohol Swab)
  • Glass slides
  • Gram stain (Crystal violet, Gram’s Iodine[Mordant], Acetone[Decolouriser], Safranin)
  • Tubes of pre-prepared 1mL saline
  • Tubes of pre-prepared 200µL rabbit plasma

For this posting, I am focusing more on the sub-culturing of blood onto plates. For more information on the BACTEC machine and how it works, refer to BMTjournal (Boon Ching’s Posting on Blood Culture). Linked with permission from Boon Ching.

Basically, the flow would be, BACTEC™ machine THEN sub-culturing of positive vials.



When the BACTEC machine detects a positive sample, the positive vial (which contains blood and can either be under Aerobic or Anaerobic conditions) is taken out. This is done through the scanning of a barcode at the door of the machine to indicate to the machine that you are removing a vial, followed by the scanning of the vial’s barcode.

The vials have 2 barcodes; one barcode is the original pre-generated BACTEC™ barcode while the other barcode is a pre-generated barcode that is provided by the lab. The laboratory barcodes comes in sets of 2. One of the pair is pasted onto the bottle and the other is pasted onto the request forms.

Following the removal of the vial, the request form is singled out, and qualified staff with access to the Laboratory Information System (LIS) will flag the vial as positive.

BACTEC machine interior

Barcodes located at the door of the machine. These are scanned to tell the machine whether you are adding vials or whether you are removing positive or negative vials


After flagging, 4 labels identical to the vial’s lab barcode numbers are generated. Each of the labels is pasted onto BAP, MAC, glass slide and 1mL saline tube. For an Anaerobic vial, an additional label is generated, with an ‘A’ after the number and this is to be pasted onto a CDC plate. (e.g. DB43210 for the 4 labels, and DB43210A for the Anaerobic plate)

Before anything is done, the cover of the vial and the glass slide is wiped with alcohol swabs. The glass slide is marked with a circle to indicate later on, the area of the drop of blood.

All of the materials, including the vial, are placed in a Biosafety Cabinet (BSC), and the whole process of sub-culturing is done within the BSC. This is to prevent the accidental exposure to aerosols from the blood.

A venting unit is attached to the vial, which would allow the blood to be slowly dripped out for testing. Firstly, 1 drop of blood is dripped each on BAP and MAC. For an Anaerobic vial, an additional drop of blood is dripped onto a CDC plate. Next, an inoculum with 6-fold dilution is obtained by adding 8 drops of blood to the tube of 1mL saline (8drops is roughly 0.2mL). Lastly, 1 drop of blood is dropped onto a glass slide and the drop is spread with an inoculating loop within the pre-indicated circle. Sometime 2 different specimens can be dropped on the same slide to avoid wastage.

The BAP, MAC and CDC (if used) are to be streaked later on after the drop of blood has partially dried. Furthermore, the BAP is streaked with Staphylococcus aureus. BAP and MAC are incubated at 35° in CO2 for 24 hours whereas the CDC is incubated under anaerobic conditions at 35° for 48 hours before reading. The inoculum (saline with 8 drops of blood) is passed to the investigation lab to be further tested. The blood on the glass slide is allowed to dry on a hotplate within the BSC, before it is gram stained.

Method for streaking
(Also shows the
S. aureus streaks done on BAP plates)

BAP that have been incubated at 35° in CO2 for 24 hours

Slides for Gram Stain (Left: Single slide, Right 2 specimens on one slide)

Gram Staining

  • Stain with Crystal Violet for 1min
  • Flush with water
  • Counter-stain with Gram’s Iodine for 1 min
  • Flush with water
  • Decolourise with Acetone for 2 sec
  • Flush with water
  • Stain with Safranin for 1 min
  • Blot dry

After staining, a presumptive preliminary identification of the organism is done on the basis of the organism being either gram positive or negative or whether it is either a bacilli or cocci. This preliminary identification is crucial for doctors as it allows them to start the patient on the relevant antibiotics.

Direct Tube Coagulase Test

This is done for Gram Positive Cocci (GPC) as a confirmatory test to check whether the GPC is S. aureus. 60 µL of blood (about 3 drops) is added to 200µL rabbit plasma and incubated for 4 hours to overnight.

As S. Aureus is capable of coagulating rabbit plasma, a positive coagulase test would suggest that the GPC is S.Aureus. Further tests are done in the investigation lab, to confirm the identification of S. Aureus.

Thats all for this posting, feel free to ask me questions. Images posted here were taken with permission from laboratory supervisor. He actually encourages us to take photos because he believes that visuals are more important in learning therefore naturally me and the others have been taking pictures non-stop, a fair bit of which is totally unrelated to learning, haha. Anyways, lets all enjoy the rest of our remaining 17 weeks of SIP :) .


Anonymous said...

hey bul* =)

what's the difference between the anaerobic and aerobic slides? are there any?

phuiyuen, TG02

J.A.M.M.Y.S said...

Answer for the first most anxious student :)

Usually for a single patient, 2 vials are taken, one Anaerobic and one Aerobic. These is done to check for the growth of organisms under either conditions.

Therefore, on an aerobic slide or plate, only aerobic organisms can multiply and can be seen. And on an anaerobic slide/plate, only anaerobic organisms can multiply and can be seen.


royal physicians said...

heya azhar....nice picture of the culture..aniwei got 2 qn to ask u....y is it that we have to wait for the blood to partially dry before streaking..and the second qn is if i'm not school we will use 70% ethanol to decolourise but here u use there any difference/which is better??
tt's all..njoy ur SIP.....

nur zahirah tg02

royal physicians said...

hello hello!! Ahhh. u r dealing with blood samples! totally cool =)

Anyway, i have the same question as Zahirah actually =S Why must the blood be partially dry?

Also, for anaerobic vial, why do u need an additional drop of blood onto the CDC plate?


J.A.M.M.Y.S said...

Congrats on being the 2nd and 3rd earlybirds...

Answer for Zahirah and Kang Ting:

Why the blood has to be partially dry?

After streaking, we keep and incubate the plates in an upside down position to prevent any water vapour condensation that forms to drop onto the medium(flashes of BMIC/MMIC anyone?).

Therefore in this position, if the drop of blood is still fluid, the blood may actually drip onto the cover or even worse, during handling of the plates, the blood can flow all over the plate thus preventing single colonies from forming.

Answer for Zahirah:

Why use acetone instead of 70% ethanol?

The main reason is that Acetone decolourises faster than 70% ethanol. The decolourising step is a crucial step in the retention or loss of Crystal Violet stain of the cell wall.

If a gram negative organism is poorly decolourised or if the slide is too quickly washed off before the stain can be decolourised, it would retain the Crystal Violet stain and thus falsely show as a gram positive organism.

Therefore the choice of decolouriser is important. If we use Acetone, we can roughly wash off the acetone straightaway. If we use 70% ethanol and wash it off too fast, and there would be a false result.

Answer for Kang Ting:

Why an additional drop of blood onto the CDC plate?

We put an additional drop of blood on CDC for anaerobic vials because we want to check for the growth of anaerobic organisms. Which would otherwise not show on the BAP and MAC plates incubated in CO2.

I hope I answered your queries and sorry for the long answers.


VASTYJ said...

hey azhar!!

why streak S. aureus together with the blood sample on BAP plate and why incubate in CO2?

and my lab also uses glass slides with 2 to 3 smears for gram staining. for 3 smears, a gram +ve is smeared on one side, gram -ve on another side and the sample in the middle. it is to compare the sample with those 2 to see whether it is gram +ve or -ve cuz sometimes staining may not be good.

Ying Ying

ALsubs said...

Hey Azhar,
I always see 2 glass bottles of blood collected for each patient and one will say aerobic n the other will say anaerobic. So, my question is why do they have to collected two bottles of blood for blood culture? and what is inside the bottles other than blood of patient?


first6weeks said...
This comment has been removed by the author.
first6weeks said...

Hi Azhar, Desmond here.

You're the only one who has had the opportunity to experience working in 2 different laboratories, albeit similar (both microbiology), I would like to know roughly what are the differences in lab safety regarding how you handle samples.

I would imagine that in Food and Water Microbiology, you dealth with food samples, whereas now you are dealing with blood. Therefore are there more precautions taken?

Desmond Heng

VASTYJ said...

How is DB? Lol, ok 1 qn ah, what is the principle behind the coagulase test?

Andre, TG01

J.A.M.M.Y.S said...

Hellooo Ying ying,

Miss you guys (Jeremy and you) at the Food and Water Micro Lab...

anyways i finally got your answer

Answer for Ying ying

Why is S. aureus streaked together with the blood sample on BAP?

The short answer is that it is to detect Haemophilus Influenzae.

The explanation for this is that the S. aureus streak provides a necessary v factor which together with an x factor found in BAP would allow Haemophilus Influenzae to multiply.

Therefore without this S. aureues streak, if there is H. Influenzae in the blood, it would not multiply and thus cannot be detected.

Why incubate in C02?

It also has to do with H. Influenzae detection.

Certain microorganisms such as H. Influenzae grow well in CO but not as well in O2. Whereas some can grow in both CO2 and O2.

Therefore, to detect microorganisms such as H. Influenzae better, we incubate in CO2


J.A.M.M.Y.S said...

Hello Sally, im wondering where you are attached. I am guessing perhaps at Haematology Lab? Anyways back to the point.

Answer for Sally

Why collect two bottles of blood for blood culture?
What is inside the bottles other than blood of patient?

These two bottles essentially contains some slight difference in content.

The Bactec/F Plus Aerobic and Bactec/F Plus Anaerobic Media contains essentiall Soybean-Casein Digest Broth with resin and 0.05% w/v (weight per volume) sodium polyanethol sulfonate (SPS).

All Bactec media are dispensed with added CO2. Anaerobic media are prereduced and dispensed with CO2 and N2

The Soybean-Casein Digest Broth and 0.05% w/v SPS basically contains the necessary nutrients for the bacteria to multiply

Anerobic media are prereduced and also contains N2, therefore it meets different conditions which would allow certain bacteria to multiply that would otherwise not be able to do so in the aerobic media


J.A.M.M.Y.S said...

Hey Desmond, interesting question.

In the Food and Water Micro Lab, we dealt with food samples from clients to check the quality of their food. These samples are almost always clean and meets government standards.

Since we do not deal with patient samples, which may very likely contain bacteria , we only need to take basic precautions. Such precatuions includes aseptic techniques using a bunsen burner.

In the Diagnostic Bacteriology Labs, there are potentially pathogenic bacteria present. Therefore, the greatest precautions are taken, such as handling blood culture in a Biosafety Cabinet to prevent inhalation of aerosols.

However it cannot be assumed that there is less lab safety precautions.

These 2 labs deal with different samples, as such, they therefore have to meet different safety requirements


J.A.M.M.Y.S said...

Hi Andre

The principle behind the coagulase test?


Coagulase is an enzyme produced by Staphylococcus aureus.

It reacts with prothrombin in the blood. The resulting complex is called staphylothrombin, which causes blood to clot by converting fibrinogen to fibrin.

The enzyme is tightly bound to the surface of the bacteria S. aureus and can coat its surface with fibrin upon contact with blood.

The coagulase test is used to differentiate Staphylococcus aureus from the other species of Staphylococcus. If the test is positive (coagulation of rabbit serum), the suspected colony is S. aureus. If negative the plasma remains liquid and thus S. aureus can be ruled out.


The Lab Freaks said...

Hi Azhar,

I'll be posted to the microbiology section at the end of the month and I'm quite turned off by it because while doing order entry, I sent alot of stool samples there.

So anyway, my question is actually not really related to your post (hahahahahahahahahahahahahaha.)but more on your experience being in micro lab (ie is it smelly? I'm so scared it will stink coz we don't get masks!!!!)

(you can probably guess its me.)

Kent said...


Was just wondering what else does the Bactec machine do (i'm guessing maybe sample incubation? =P) and how it identifies positive samples?

Kent Lieow

J.A.M.M.Y.S said...

Yes of course I can tell its you, Sharifah

The thing is, the processing area of the micro lab aint that smelly.

Stool cultures are done in Biosafety Cabinets (BSC) which ensures that the fume would not escape provided you follow the instructions of the BSC.

The urine culture however is soo much more different. This is the 'station' that receives the most samples in a day, more than 100 a day. And the plates are streaked on a normal lab bench.

The investigation labs are the most smelly. This is because the bacteria have all formed colonies and are are producing all sorts of 'wonderful' smells. Hahaha.

Good luck for your posting, I'm sure you'll get used to it...eventually...


J.A.M.M.Y.S said...

Hey Kent,

Actually that question is more related to Boon Ching's blog, but since you're already here i'll just explain it.

The BACTEC vials are arranged in racks and are continuously incubated aerobically at 35 degrees celsius and are agitated(rocked slowly) throughout the standard 5 days protocol.

If microorganisms are present in the BACTEC vial, CO2 will be produced when the organisms metabolize the substrates

All the vials has a sensor at the bottom that fluoresces. The increased CO2 are monitored by the BACTEC machine through the increase in fluorescence of the vial sensor.

Analysis of the rate and amount of CO2 increase enables the BACTEC fluorescent series instrument to determine if the vial is positive (contains viable organisms)

Hope that helps


ALsubs said...


Most of the quests that i wanted to ask were already asked by the rest.. So finally i have only one question to ask you. With this test, wat kinda diagnosis or interpretation can u make based on the results obtained during ur SIP