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newSchoolWorks.com › VoTechWorks › Agriculture, Environment & Public Service › Aquaculture & Marine Science Tech › Hatchery Operations: Broodstock to Juvenile Production › Lesson 7
AGR-AQUA50-04 · Lesson 7 · 16 clock hours · lab

Live Feed Culture: Microalgae Production

Objective. Students will maintain a continuous microalgae culture of at least one species (e.g., Nannochloropsis, Tetraselmis, or Isochrysis), scale up from a flask to a carboy, and use a hemocytometer or spectrophotometer to estimate cell density against a target harvest density.

⚠ Pending professional review

This lesson, its worked examples and its practice questions has not yet been verified by a licensed professional in the field or by a certification or approval body. It is a study draft and may contain errors. Do not use it as instructions for work on a job site or on live equipment. Always follow your instructor, your supervising professional, and current official references and regulations.

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Why Microalgae Matter in a Hatchery

Microalgae are the foundation of the marine food web and the first live feed used in most hatcheries. Larvae of oysters, clams, shrimp, and many finfish cannot digest formulated diets in their earliest stages; they need living algal cells that are nutritionally complete and the right size. Your job as a hatchery technician is to grow these cultures reliably, keep them clean, and know when they are ready to harvest. The three species you are most likely to work with are Nannochloropsis (small, high in EPA fatty acids, used for rotifers and early bivalve larvae), Tetraselmis (larger, robust, and nutritionally broad), and Isochrysis (rich in DHA, essential for finfish larvae).

Growth Stages and Scale-Up Logic

Microalgae growth follows a predictable curve: a short lag phase when cells adjust to new conditions, exponential (log) phase when cells divide fastest, a stationary phase as nutrients are consumed, and a crash phase when the culture declines. You want to harvest during late exponential or early stationary phase, when cell density and nutritional quality are both high.

Scale-up moves cultures from smaller vessels to larger ones in steps — typically from a 125 mL flask to a 2 L carboy, then to larger bags or tanks. You add a volume of dense starter culture (the inoculum) to a larger volume of fresh medium at roughly a 1:5 to 1:10 ratio. The new vessel must be pre-sterilized so the incoming cells are not immediately outcompeted or killed by contaminants.

Aseptic Technique

Every transfer is a contamination risk. Aseptic technique means you work in a way that excludes unwanted microorganisms:

Safety note: Autoclaves operate at high pressure and temperature. Always wear heat-resistant gloves and eye protection when loading, unloading, or cracking the autoclave door. Wait until pressure returns to zero before opening. If a pressure cooker is used instead, follow the same wait-for-depressurization rule.

Growth Conditions

Microalgae need light, CO₂ or aeration (to supply carbon and keep cells suspended), and nutrients.

Light: Cool-white fluorescent or LED grow lights positioned close to the vessel. Most hatchery species grow well on a 16-hour light / 8-hour dark photoperiod, though continuous light is used in some facilities.

Aeration: Gentle but continuous air bubbling prevents settling, distributes CO₂, and promotes gas exchange. Set the flow rate so the culture mixes visibly without splashing over the opening.

Medium: The most widely used formulation in marine hatcheries is f/2 medium (Guillard & Ryther), which supplies nitrate, phosphate, silicate (for diatoms), vitamins, and trace metals at defined concentrations. It is diluted into filtered, autoclaved or UV-treated seawater at the correct salinity for your species.

Measuring Cell Density

Hemocytometer Method

A hemocytometer is a thick glass slide with two counting chambers, each engraved with a grid. The most common type (Neubauer improved) has a known chamber depth of 0.1 mm. Each large square has an area of 1 mm², so the volume over one large square is 0.1 mm³ = 1 × 10⁻⁴ mL.

Formula:

Cells per mL = (average count per large square) × 10⁴ × dilution factor

Count at least five large squares and average them. If the culture is very dense, dilute it (for example, 1:10 in seawater) before counting, then multiply by the dilution factor.

Spectrophotometer Method

A spectrophotometer measures how much light a suspension absorbs at a specific wavelength (often 680 nm for chlorophyll-containing cells). The absorbance reading — optical density (OD) — is compared to a calibration curve built from samples of known cell density. The curve is species-specific because cell size and pigment content differ between species. Read the OD, find the corresponding cell density on your calibration chart, and correct for any dilution you made.

Identifying Contamination

A healthy culture looks evenly colored (green, golden-brown, or gray-green depending on species) and smells neutral or faintly of the sea. Signs of contamination include:

Corrective action: A bacterially or protist-contaminated culture cannot be cleaned in place reliably. The recommended response is to autoclave the contaminated vessel to kill everything, then restart from a verified clean stock. UV treatment of incoming seawater is a preventive measure, not a cure for an already-infected culture. Document every contamination event in the live-feed log with date, species, likely source, and action taken — this record is how your facility improves its protocols over time.

Personal Protective Equipment

Wear a lab coat or apron, nitrile gloves, and safety glasses at the bench. When handling concentrated acids or bases used to adjust medium pH, add a face shield. Remove gloves before touching shared surfaces such as door handles or computer keyboards.

Key terms8

InoculumA small volume of dense, healthy starter culture added to a larger volume of fresh medium to begin a new culture vessel.
f/2 MediumA standard seawater-based nutrient formulation (Guillard & Ryther) supplying nitrate, phosphate, trace metals, and vitamins for growing marine microalgae in hatcheries.
Aseptic TechniqueA set of practices — sterilizing equipment, working near a flame or in a clean-air hood, minimizing exposure time — that prevent microbial contamination during culture transfers.
HemocytometerA precision glass counting slide with an engraved grid and known chamber depth used to count cells in a small sample and calculate cells per mL.
Optical Density (OD)A spectrophotometer reading of how much light a culture absorbs at a set wavelength; used with a species-specific calibration curve to estimate cell density without counting individual cells.
Exponential (Log) PhaseThe growth stage when algal cells are dividing at their maximum rate; cultures are typically harvested at the end of this phase or at the beginning of stationary phase.
Calibration CurveA graph or table relating OD readings to known cell densities for a specific algal species, allowing technicians to convert a spectrophotometer reading into a cell count estimate.
Contamination (bacterial or protist)The unwanted presence of bacteria or protozoa in an algal culture, identified visually and microscopically, that can crash the culture and requires autoclaving and restarting from clean stock.

Worked examples4

1. You count cells in five large squares of a Neubauer improved hemocytometer. Your counts are 42, 38, 45, 40, and 35. The sample was diluted 1:10 before counting. What is the estimated cell density in the original culture?
  1. Step 1 — Average the five counts: (42 + 38 + 45 + 40 + 35) ÷ 5 = 200 ÷ 5 = 40 cells per large square.
  2. Step 2 — Apply the hemocytometer conversion factor: 40 × 10,000 = 400,000 cells per mL in the diluted sample.
  3. Step 3 — Correct for the 1:10 dilution: 400,000 × 10 = 4,000,000 cells per mL in the original culture.
Answer: 4.0 × 10⁶ cells per mL (4,000,000 cells/mL) in the original culture.
2. Your target harvest density for Nannochloropsis is 20 × 10⁶ cells per mL. Your hemocytometer count (no dilution, average of five squares = 55 cells per square) gives a certain density. Is the culture ready to harvest?
  1. Step 1 — Calculate current density: 55 × 10,000 = 550,000 cells per mL = 0.55 × 10⁶ cells per mL.
  2. Step 2 — Compare to target: 0.55 × 10⁶ cells per mL is far below the 20 × 10⁶ cells per mL target.
  3. Step 3 — Decision: the culture is not ready to harvest; continue growing and re-count in one to two days.
Answer: Not ready. Current density is approximately 5.5 × 10⁵ cells/mL, which is about 2.75% of the 20 × 10⁶ cells/mL harvest target.
3. You are scaling up from a 125 mL flask to a 2 L carboy using a 1:10 inoculum-to-total-volume ratio. How many mL of starter culture do you need, and how many mL of fresh f/2 medium do you add?
  1. Step 1 — Determine total target volume: 2,000 mL.
  2. Step 2 — Calculate inoculum volume at a 1:10 ratio: 2,000 ÷ 10 = 200 mL of starter culture.
  3. Step 3 — Calculate medium volume: 2,000 − 200 = 1,800 mL of fresh f/2 medium.
Answer: Add 200 mL of starter culture and 1,800 mL of fresh f/2 medium to fill the 2 L carboy.
4. A spectrophotometer reads OD₆₈₀ = 0.45 for an Isochrysis sample. The calibration curve equation for this species is: cell density (cells/mL) = OD₆₈₀ × 8 × 10⁶. The sample was diluted 1:2 before reading. What is the cell density in the original culture?
  1. Step 1 — Use the calibration equation with the OD reading: 0.45 × 8 × 10⁶ = 3.6 × 10⁶ cells per mL in the diluted sample.
  2. Step 2 — Correct for the 1:2 dilution: 3.6 × 10⁶ × 2 = 7.2 × 10⁶ cells per mL in the original culture.
Answer: 7.2 × 10⁶ cells per mL in the original Isochrysis culture.

The math in this lesson2

Learners apply the ×10,000 factor and 1:10 dilution to report cells/mL in scientific notation.
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Brush up in the math halls at newSchoolWorks.com:
In class: Culture transfer and scale-up: following aseptic technique, students transfer a flask culture into a two-liter carboy with f/2 or equivalent medium, set aeration and lighting parameters, label with species, transfer date, and their initials, and record in the live-feed log. · Cell density assessment: students prepare a hemocytometer slide, count cells in five grid squares, calculate cells-per-mL using the hemocytometer formula, and compare to the target harvest density; or use a spectrophotometer and a species-specific OD-to-cell-density calibration curve to estimate density. · Contamination identification exercise: instructor provides three flasks—one clean culture, one with bacterial contamination, one with protist contamination—and students examine each under a microscope, document observations, and recommend corrective action (autoclave and restart, or UV treatment) for the contaminated flasks.
Materials: Starter microalgae cultures, f/2 or equivalent liquid medium, 125 mL and 2 L carboys, aeration tubing, cool-white fluorescent or LED grow lights, autoclave or pressure cooker, hemocytometer with cover slips, compound microscope, spectrophotometer (optional), live-feed log, aseptic transfer hood or laminar-flow if available
Check: Culture transfer completed using aseptic technique with no cross-contamination (verified by instructor observation at transfer); cell density estimate within 20% of instructor reference count; contamination identification correct for all three flasks — 100% correct on contamination IDs required.

Practice check8

Answer each question, then check it. Nothing is recorded; this is your own practice.

Hatchery Operations / Live Feed Production
1. What is the primary purpose of autoclaving glassware and medium before a culture transfer?
Hatchery Operations / Cell Density Measurement
2. You average 60 cells per large square on a hemocytometer (Neubauer improved) from an undiluted sample. What is the cell density in cells per mL?
Hatchery Operations / Algal Culture Management
3. Which growth phase represents the best time to harvest microalgae for feeding larvae in a hatchery?
Hatchery Operations / Contamination Identification
4. Under the microscope, you observe large, actively moving single-celled organisms in a Nannochloropsis culture that has crashed rapidly. The most likely cause is:
Hatchery Operations / Contamination Control
5. A contaminated microalgal culture has been identified. What is the recommended corrective action?
Hatchery Operations / Cell Density Measurement
6. When using a spectrophotometer to estimate algal cell density, why must you use a species-specific calibration curve?
Laboratory Safety / PPE
7. Which personal protective equipment is required when working with an autoclave in the hatchery lab?
Hatchery Operations / Algal Culture Troubleshooting
8. You scale up a Tetraselmis culture to a 2 L carboy at a 1:10 inoculum-to-total-volume ratio. Two days later, the culture appears pale and the OD reading is lower than expected. Aeration is adequate and lighting is on schedule. What is the most likely cause?
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