Why was API RP 38 developed?

Water injection has been used for decades to maintain reservoir pressure and improve oil recovery. Once large volumes of water are collected, treated and reinjected, microorganisms become an operational concern.

Microbial growth in an injection-water system can contribute to:

• biofilm formation;
• filter and formation plugging;
• loss of injectivity;
• hydrogen sulfide generation and reservoir souring;
• deposit formation;
• chemical degradation;
• and microbiologically influenced corrosion.

API RP 38 emerged in this historical context. The objective was practical: provide the oil industry with a repeatable approach for examining the microbiological quality of water used in water-flood and subsurface injection systems.

What did API RP 38 entail?

API RP 38 should be understood primarily as a culture-based oilfield water microbiology practice.

Rather than directly counting every microorganism in an injection-water sample, the approach depends on microorganisms being transferred into culture media and then reproducing under the conditions provided by those media.

The practical workflow that became associated with API RP 38 can be summarized as follows:

For sulfate reducers, a positive culture is commonly recognized through sulfide production and subsequent formation of black iron sulfide.

For general bacterial culture systems, growth may instead be evaluated through turbidity or another medium-specific reaction.

The fundamental principle is therefore growth-dependent enumeration. No growth means that no qualifying growth was detected under the conditions used; it does not prove that the original water contained no microorganisms.

For a broader explanation of oilfield culture workflows, read MPN Protocols, PBS Buffer and Culture Media for Oilfield Microbiology.

What is API RP-38 SRB medium?

In modern oilfield terminology, “API medium” or “API RP-38 medium” usually refers to an anaerobic culture medium intended to recover sulfate-reducing bacteria or sulfate-reducing microorganisms.

Commercial and research formulations carrying the API RP-38 name are not necessarily identical. This is important because the original API publication is historical, while later suppliers and laboratories have adapted media for salinity, storage, redox stability and field use.

An API-type SRB medium commonly provides the following functional components:

Why is an iron nail associated with API RP-38 bottles?

The traditional visual image of API SRB medium is a clear culture vial containing an iron nail.

Modern commercial API-type instructions describe the nail as part of the system used to maintain suitable reducing conditions and provide iron for the visible sulfide reaction. Other formulations use dissolved ferrous iron instead of, or in addition to, solid iron.

This is one reason why laboratories should not assume that every bottle labelled “API” represents exactly the same medium.

For a detailed comparison, read Postgate B, API RP-38 and Starkey Media: What Is the Difference?.

How does the API RP 38 serial-dilution approach work?

The practical oilfield method is often called the serial dilution, serial extinction or bug-bottle technique.

In a common commercial implementation:

1. A defined volume of the original sample is injected into the first vial.
2. The vial is mixed.
3. A defined aliquot from the first vial is transferred into the second vial.
4. The process is repeated through a series of progressively more dilute bottles.
5. The cultures are incubated.
6. Positive and negative bottles are recorded.

If the transfer scheme creates tenfold steps, each successive bottle contains approximately one tenth of the concentration represented by the previous bottle.

A result is then often expressed according to the last or highest dilution showing a positive reaction.

For this distinction, see MPN Calculator for Oilfield Microbiology and What Does “Less Than the Detection Limit” Mean in an MPN Test?.

Why does an API SRB bottle turn black?

Sulfate-reducing microorganisms can use sulfate as an electron acceptor and produce reduced sulfur compounds including sulfide.

When sulfide contacts ferrous iron in the culture system, poorly soluble black iron sulfide can form.

The resulting black precipitate provides a convenient visual endpoint.

However, the chemical reaction itself does not prove that the sulfide was produced after inoculation.

Oilfield water may already contain:

• dissolved H₂S or HS⁻;
• fine iron-sulfide particles;
• black corrosion products;
• or sulfide released from deposits.

If these enter an API culture vial, rapid blackening can occur without new microbial growth.

Commercial API-type instructions therefore warn users to distinguish immediate sample-derived blackening from progressive blackening that develops during incubation.

Read Can Sulfide Cause a False-Positive SRB Test? for the detailed interpretation.

How long is API-type SRB culture incubated?

Long incubation is another characteristic associated with conventional API-style oilfield culture.

Current commercial instructions explicitly referring to API RP-38-type SRB media commonly use an incubation period of up to 28 days for sulfate-reducing cultures.

For example, Biotechnology Solutions' commercial API serial-dilution procedure specifies 28 days for SRB media. OFITE's bacteria-test instructions likewise state that API RP 38 recommends a 28-day incubation for anaerobic vials.

A strongly positive bottle can blacken much earlier. The long endpoint is especially important when deciding whether a bottle that remains unchanged can finally be classified as negative under the selected procedure.

For the wider commercial comparison, see How Long Should You Incubate SRB and APB Test Bottles?.

What does an API RP 38 result actually measure?

This is the most important point for modern interpretation.

An API RP-38-style culture result does not count every sulfate-reducing microorganism present in the original oilfield sample.

It detects the fraction that can successfully pass through all of the following selection steps:

• the organisms must be present in the small aliquot that enters the culture;
• they must survive sample collection and handling;
• they must tolerate oxygen exposure during transfer;
• they must tolerate the medium salinity;
• they must grow at the selected incubation temperature;
• they must utilize the nutrients and electron donor supplied by the medium;
• they must grow within the selected incubation period;
• and they must produce enough sulfide to generate the defined positive endpoint.

The result is therefore more accurately described as:

This explains why changing culture medium can change the reported SRB count even when the field sample itself has not changed.

See Why Do MPN Results Differ Between Laboratories?.

How does API RP 38 compare with more recent standards?

API RP 38 is sometimes described as though it was simply replaced by one newer standard. That is too simplistic.

More recent AMPP standards address different layers of the microbiology and MIC problem.

AMPP TM0194: the more relevant culture-monitoring reference

AMPP currently lists TM0194-2014, Field Monitoring of Bacterial Growth in Oil and Gas Systems, among its MIC-related standards.

For a new programme based on field culture and serial dilution, TM0194 is therefore a much more appropriate starting reference than a withdrawn API publication.

It is important, however, not to interpret TM0194 as proof of MIC. Its function is microbial monitoring.

AMPP TM0212: when the question is MIC rather than microbial count

AMPP TM0212 addresses the detection, testing and evaluation of MIC on internal pipeline surfaces.

This difference in scope is critical.

An API RP-38-style result may tell you that culturable sulfate reducers were detected in a water sample. It does not establish that those microorganisms:

• were attached to the corroding steel;
• were active at the damage location;
• were responsible for the observed morphology;
• or accelerated the measured corrosion rate.

AMPP's public MIC guidance emphasizes the need to combine biological, chemical, metallurgical and operational lines of evidence.

Read Why a Produced-Water Sample Alone Cannot Confirm or Exclude MIC.

AMPP TM21465: bringing molecular microbiology into the standards framework

API RP 38 was created decades before qPCR, modern sequencing and today's molecular microbial ecology became practical oilfield tools.

AMPP TM21465-2024 addresses molecular microbiological methods through guidance on sample handling, preservation, laboratory processing and data analysis.

This represents a fundamentally different measurement philosophy.

Culture asks:

Which microorganisms could grow under the conditions supplied by this bottle?

Molecular testing can instead ask:

Was a defined organism, microbial group or functional gene present in the material that was sampled?

Neither answer should be confused with proof of MIC causation.

Is API RP 38 still relevant?

Yes—but mainly as a historical and legacy method, not as the preferred current standard for building a new MIC monitoring programme.

What are the main limitations of API RP 38 for modern MIC monitoring?

1. It is a withdrawn standard

The most basic limitation is formal status. API lists publication 38 in its withdrawn historical-publications catalogue.

A new project should therefore not describe itself as using a current API standard simply because it uses an API-type SRB medium.

2. Culture detects recoverable growth, not the complete microbial population

Microbial communities in oilfield systems are diverse. Many organisms do not grow efficiently in one standardized lactate-based bottle.

A clear API culture therefore does not mean that the sample contained no sulfate-reducing microorganisms.

See Why Does an SRB Bottle Stay Clear Even When Sulfide Is Present?.

3. Medium composition introduces strong selection

The electron donor, mineral composition, redox chemistry, temperature and salinity select the organisms that can recover.

Changing from API to Modified Postgate B can change the reported concentration without any real increase or decrease in the field population.

4. Planktonic water may not represent the corroding surface

API RP 38 originated around injection-water analysis.

MIC, however, is usually a surface-associated process involving biofilms, deposits and local electrochemistry.

Organisms in bulk water may differ from those inside a deposit or directly attached to a corrosion pit.

5. Sulfide can interfere with the visual endpoint

Blackening is useful but not perfectly specific for new microbial growth. Pre-existing sulfide and black corrosion solids can interfere.

6. Long incubation reduces operational speed

A final culture endpoint of several weeks is poorly matched to some operational decisions, particularly when operators are evaluating a rapidly changing treatment response.

7. A positive SRB culture does not quantify corrosion risk

There is no universal API RP-38 concentration above which MIC is automatically active or severe.

Read What Is a High SRB Count in Oil and Gas?.

What can API RP 38 not tell you about modern MIC mechanisms?

Our understanding of microbiologically influenced corrosion has changed significantly since the 1950s and 1970s.

Historically, SRB monitoring often treated sulfate reducers as one broad operational risk group, with sulfide production as the main biological indicator.

We now know that this is incomplete.

Different sulfate-reducing microorganisms can interact with steel very differently. Relevant processes can include:

• production of sulfide;
• formation of iron-sulfide deposits;
• consumption of hydrogen;
• biofilm-driven concentration cells;
• metabolic interactions with fermenters and methanogens;
• and, for selected organisms, extracellular electron-transfer mechanisms.

A broad API RP-38 SRB culture cannot distinguish these mechanisms.

The same argument applies to methanogenic Archaea. A traditional SRB bottle was never designed to characterize methanogenic MIC or archaeal extracellular electron-transfer pathways.

API RP 38 versus qPCR: are they competing methods?

Not directly. They measure different biological properties.

This is why culture and qPCR can be complementary rather than interchangeable.

How should an operator modernize a legacy API RP 38 programme?

Replacing a historical method overnight can destroy useful trend information.

If an asset has ten or twenty years of API-medium results, that historical baseline has value even if the method itself is no longer state of the art.

A controlled transition is therefore preferable.

Step 1: document the actual legacy method

Do not write only “API RP 38”. Record:

• supplier;
• media formulation or product identifier;
• salinity;
• bottle volume;
• dilution scheme;
• sample volume;
• incubation temperature;
• incubation period;
• positive-reaction criteria;
• and reporting convention.

Step 2: run the old and new methods in parallel

For a defined transition period, analyze representative samples using both the historical API-type method and the proposed replacement or supplementary method.

This creates a bridging dataset.

Step 3: include different microbial conditions

The comparison should include more than clean, untreated water.

Where possible, include:

• high and low microbial loads;
• pre- and post-biocide samples;
• different salinities;
• different temperatures;
• produced water;
• injection water;
• deposits or surface-associated samples where relevant;
• and samples with known sulfide interference.

Step 4: do not transfer old action limits automatically

An action level of 10³ organisms/mL developed using API medium cannot automatically be applied to a more sensitive Postgate formulation or to a qPCR result.

The measurement property has changed.

Step 5: separate routine monitoring from MIC diagnosis

A routine water-monitoring programme and an MIC root-cause investigation should not be treated as the same exercise.

Routine trending may use water samples and consistent operational assays. A suspected MIC failure should include surface-associated samples, corrosion morphology, deposits, chemistry and operating history.

Step 6: add molecular methods for questions culture cannot answer

Targeted qPCR can add rapid information on selected taxonomic groups and functional genes without waiting for culture growth.

Periodic sequencing can be useful when the objective is to check whether the routine target panel still represents the broader microbial community.

Does a newer standard make historical API RP 38 data useless?

No.

A 20-year trend generated with one stable culture method can contain important operational information. Abruptly replacing it with another method can make apparent changes difficult to interpret.

The key is to distinguish method validity for current use from historical value for trend interpretation.

Old API-type data can remain useful when:

• the culture medium is known;
• sampling locations remained consistent;
• incubation conditions were stable;
• the reporting convention did not change;
• and the limitations of the culture method are understood.

Historical data become much less defensible when records simply state “SRB count” without identifying the test system.

Frequently asked questions

Sources and further reading

1. American Petroleum Institute. Historical Publications Catalogue . API lists withdrawn publications and records publication 38, Biological Analysis of Water-Flood Injection Waters, first edition 1959 through third edition 1975.
2. American Petroleum Institute. API Standards: International Usage Report, 2025 Edition . Includes an example of API RP 38, Recommended Practice for Biological Analysis of Subsurface Injection Waters, remaining referenced in a contemporary legacy service catalogue.
3. AMPP. Microbiologically Influenced Corrosion resources and standards . Lists TM0194-2014 and TM0212-2018 and describes the need for multiple lines of evidence in MIC assessment.
4. AMPP. TM0194-2014, Field Monitoring of Bacterial Growth in Oil and Gas Systems. Consult the licensed current publication for normative procedures and requirements.
5. AMPP. TM0212-2018, Detection, Testing, and Evaluation of Microbiologically Influenced Corrosion on Internal Surfaces of Pipelines.
6. AMPP. TM21465-2024, Molecular Microbiological Methods—Sample Handling and Laboratory Processing .
7. Biotechnology Solutions. Serial Dilution Instructions (API) . Commercial API-RP38-type SRB medium instructions illustrating iron-nail media, dilution procedure, sulfide interference and 28-day SRB incubation.
8. OFITE. Bacteria Test Kit for Aerobic and Anaerobic Bacteria. Commercial field instructions referring to API RP 38 incubation periods and oilfield serial-dilution culture.
9. MICBUSTERS. Postgate B, API RP-38 and Starkey Media: What Is the Difference?
10. MICBUSTERS. MPN Protocols, PBS Buffer and Culture Media for Oilfield Microbiology

Standards note: This article explains the historical and technical context of API RP 38 and the public scope of newer standards. It does not reproduce API RP 38, AMPP TM0194, TM0212 or TM21465 and does not establish compliance with any standard. Normative requirements should be taken from the licensed publication applicable to the project.