Glass Pipette: Types, Mohr's vs. Serological, and How to Use One Correctly

In a microbiology practical, a student is performing a tube agglutination test. She needs to transfer exactly 1 mL of patient serum to each tube in a serial dilution series. She picks up a serological pipette, fills it to the 1 mL graduation mark, dispenses, then moves through the dilution series.

At the end, the results are inconsistent: the agglutination pattern doesn't follow the expected gradient. The problem surfaces during review. She had been using a serological pipette correctly, blowing out the last drop, but she had read the meniscus from the top of the curve rather than the bottom.

Each "1 mL" delivery was actually 1.05 to 1.1 mL. Across eight tubes, the dilution factor drifted. The test had to be repeated.

Glass pipette technique comes down to two things that seem minor until they aren't: knowing which type of pipette you are holding (drain-out or blow-out), and reading the meniscus from the correct reference point. Both errors are invisible at the time they happen and only appear in the results.

Pipettes are calibrated instruments used to handle liquids. Several kinds are used in the laboratory, including the micropipette, the glass pipette, and the automated pipette.

Among those, glass pipettes are one of the liquid handling instruments carrying utmost significance due to their precision. These are useful in handling samples with volumes in the milliliter (mL) range.

Glass pipettes are of different types, and handling of glass pipettes requires an attached filler (pipette bulb or pipette filler) at the top for filling the liquids. They have uses in various sectors like chemical laboratories, biochemistry laboratories, forensic science, microbiology laboratories, etc.

Why Glass Pipettes Matter in Microbiology

The micropipette has replaced glass pipettes for most microliter-scale work. But glass pipettes remain the instrument of choice whenever volumes in the milliliter range need to be transferred, and in microbiology this is more common than students expect.

Preparing liquid culture media requires transferring 5 mL, 10 mL, or 25 mL volumes of reagents and supplements with accuracy. Serological tests such as tube agglutination, the Widal test, VDRL, and complement fixation require serial dilutions in mL volumes where a volumetric pipette delivers the precision needed.

Blood culture bottles are inoculated with defined volumes of blood (8 to 10 mL for adult bottles). Antimicrobial stock solutions are prepared in mL volumes before being diluted to µL-scale working concentrations.

In each of these contexts, choosing the wrong glass pipette type, or using the right type incorrectly, introduces a volume error that propagates through every subsequent step.

Types of Glass Pipettes

The glass pipettes generally used have three types; graduated, non-graduated, and Pasteur pipettes.

Graduated pipettes

Graduated glass pipettes have increments marked along a straight glass tube, and are also called measuring pipettes. By accuracy, they are graded Class A, Class AS, and Class B.

Class A and Class AS are the high-accuracy grades with tightly specified error limits; the two differ mainly in waiting time, with Class AS designed for faster delivery. Class B is the general-purpose grade, with error limits roughly double those of Class A.

Graduated pipettes are further classified into two types based on the graduation mark and nominal volume it can pipet; Mohr’s and serological pipette.

• Mohr's pipette: a drain-out pipette. It is a straight tube with a graduation mark at each 0.1 mL interval, and the lowest mark begins well above the tip, so the liquid below the last mark is never delivered. If the tip breaks, the pipette can no longer be used accurately, because the undelivered volume below the last mark is no longer defined.
• Serological pipette: A serological pipette is used as a blow-out pipette. It is a straight tube with a graduation mark near the tip. A slight pressing of the bulb is necessary at the end of dispensation for accuracy.

Mohr's vs. Serological Pipette

These two pipettes look almost identical. The difference is in how the last drop is handled, and confusing them introduces a consistent delivery error.

The practical rule: Before using any graduated glass pipette, check the tip. If the graduation marks stop before the tip, it is a Mohr's, so do not blow out. If the marks extend to the tip and there is a ring near the top, it is serological, so blow out completely.

You will also meet these two pipettes under other names.

1. A drain-out pipette (Mohr's) is a non-blow-out pipette: you let it drain and leave the last drop.
2. A blow-out pipette (serological) is marked with a frosted or etched band near the top, which is the standard signal that the final drop must be blown out to deliver the full volume.

Separately, pipettes carry a calibration marking: "to deliver" (TD, sometimes written "ex") means the pipette is calibrated to dispense its stated volume by draining, while "to contain" (TC, "in") means it holds that volume including the film left on the wall. Most graduated and volumetric pipettes are TD. The blow-out band and the TD or TC mark are both printed near the suction end, so check the top of the pipette before you start.

Volumetric (bulb) pipette

A volumetric pipette, also called a bulb pipette or transfer pipette, has a single calibration mark on a bulb-shaped stem and delivers one exact volume. Because it has one mark rather than a graduated scale, it is the most accurate glass pipette for its rated volume, and it is the pipette of choice when precision matters more than flexibility, for example delivering exactly 25 mL in volumetric analysis or serology.

Pasteur pipettes

Pasteur pipettes, also called droppers or eye droppers, taper to a narrow opening at the tip and are used for approximate transfer, not measured volumes. They are made in glass or plastic.

The plastic type has an integral squeeze bulb at the top; the glass type needs a separate rubber bulb (teat) to draw up liquid. A glass Pasteur pipette fitted with a rubber teat is often called a teat pipette.

Parts of Glass Pipettes

Figure: Parts of glass pipette

The parts of glass pipettes are as follows:

• Orifice of the tip: It is the pointed end of the pipette and comes in direct contact with the liquid.
• Calibration details: It is an area in the pipette above the graduation mark. It has the imprinted details about the manufacturer, volume range (measuring) or nominal volume (bulb pipette), tolerance volume, calibration temperature, standard applied for calibration, and waiting time.
• Color rings: some manufacturers add a color ring or band near the top as a code for the pipette's nominal volume.
• Suction end: It is the top part of the pipette, where the pipette filler or bulb can attach for aspirating and dispensing the liquids.
• Additional parts: The bulb pipette has a volume mark for pipetting or transferring the correct amount of liquid volume. The blow-out or serological pipette has a blow-out ring near the topmost or suction part of the pipette.

Handling of Glass Pipettes

While handling glass pipettes, one should never aspirate fluids in the laboratory from the mouth. A pipette bulb, rubber bulb, or filler is used for aspirating.

Reading the Meniscus

The meniscus is the curved surface of liquid at the graduation mark. Reading it incorrectly produces a consistent volume error on every pipetting step.

For colorless and light-colored liquids (water, saline, most reagents): Read from the bottom of the meniscus curve, the lowest point of the concave surface, with the eye level with the graduation mark. Reading from the top of the meniscus over-reads the volume.

For dark, opaque, or strongly colored liquids where the bottom of the curve cannot be seen (strong iodine, some dyes): read from the top of the visible meniscus. Mercury is a special case: it does not wet glass and forms a genuinely convex meniscus, so it too is read from the top.

Parallax error: If the eye is above or below the level of the graduation mark when reading, the mark appears to intersect the meniscus at the wrong point. Always position the eye exactly level with the graduation mark, not above and not below.

A simple habit eliminates both errors: before every pipetting step, lower your head until your eye is level with the graduation mark, then read. This takes two seconds and prevents the most common volume error in glass pipette technique.

Pipette Bulb

Figure: Pipette filler or pipette bulb Image source: Paweena.S, CC BY-SA 4.0

A pipette bulb is made up of rubber, so it is also called a rubber bulb. It is used to safely fill the pipette with liquid in the laboratory, hence named pipette filler. The generally used pipette filler are general purpose bulbs, three-valve bulbs, and thumb wheel type fillers (made of plastic).

Steps for using three valve pipette bulb:

Filling the Liquid

1. Fill the pipette with pipette filler approximately above 5 mm of the mark.
2. Then, adjust and bring the liquid to the graduated mark.
3. Set the meniscus at the mark. For colorless liquids, line up the bottom of the meniscus with the mark; for dark or opaque liquids where the bottom cannot be seen, line up the top of the meniscus with the mark.
4. Remove excess liquid from the tip for precise delivery.

Dispensing the Liquid

1. Place the tip of the pipette into the container.
2. Press the filler to dispense all the liquid.
3. Wait for a few seconds when the liquid reaches the tip (based on the company’s guidelines).
4. After the wait time, draw the pipette upwards by touching the wall to dispense the remaining liquid. Do not blow out the remaining liquid.

Common Errors in Glass Pipette Use

Uses of Glass Pipettes

The glass pipette is used across almost all science laboratories. The main fields are as follows:

• Chemistry laboratory: It is used in transferring liquids during various processes. The volumetric pipette is helpful in different tests relating to volumetric analysis.
• Pharmaceutical industry: glass pipettes are used in drug production, and to transfer measured volumes during quality control.
• Microbiology laboratory: glass pipettes transfer reagents and samples in milliliter volumes in many procedures.
• Biochemistry laboratory: The primary uses of glass pipettes in the biochemistry laboratory are as follows:
  • For preparing buffer solution.
  • To transfer the solution for conducting different biochemical tests.
  • For accurate measurements of the chemicals.

For a comparison of all pipette types used in the microbiology laboratory, see Types of Pipettes Used in the Microbiology Laboratory

How to Remember

Mohr's drains, serological blows: the easiest distinction in pipetting. Mohr's drains out, like a drain pipe where liquid flows and stops. Serological blows out, like blowing the last drop out of a straw. If you remember nothing else about glass pipettes, remember this one distinction, because it prevents the most common delivery error.

The meniscus rule: "Colorless = bottom; colored = top." For transparent liquids, read the bottom of the curve. For opaque or colored liquids, read the top. And always, without exception, get your eye level with the mark before reading.

Glass pipettes think in mL; micropipettes think in µL. If your volume has a decimal point and the unit is µL, you need a micropipette. If your volume is a whole number in mL (1 mL, 5 mL, 10 mL), glass pipettes are the right instrument. Crossing this boundary, using a glass pipette for µL work, introduces errors large enough to invalidate the result.

Key exam facts in one table

References

1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries, Part 2. 2nd edn. Cambridge University Press.
2. International Organization for Standardization (2007). ISO 835:2007. Laboratory Glassware: Graduated Pipettes. ISO.
3. Mahon CR, Lehman DC, Manuselis G (2018). Textbook of Diagnostic Microbiology. 6th edn. Elsevier.
4. Leber AL, editor (2016). Clinical Microbiology Procedures Handbook. 4th edn. ASM Press. doi:10.1128/9781683670438.CMPH